Dual cytokine fusion proteins comprising il-10

ABSTRACT

The application relates to a dual cytokine fusion protein composition, pharmaceutical composition, and/or formulation thereof comprising IL-10 or IL-10 variant molecules fused to a single chain variable fragment scaffolding system and a second cytokine, where the second cytokine is linked in the hinge region of the scFv. The application also relates to methods of using the dual cytokine fusion protein composition for treating cancer, inflammatory diseases or disorders, and immune and immune mediated diseases or disorders.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation application of U.S. patentapplication Ser. No. 17/110,104, entitled “Dual Cytokine Fusion ProteinsComprising IL-10,” filed on Dec. 2, 2020, which claims priority to U.S.Provisional Application No. 63/054,208 filed Jul. 20, 2020, thedisclosure of each is incorporated by reference in its entirety.

FIELD OF INVENTION

The present disclosure relates to the field of biotechnology, and morespecifically, to a novel dual cytokine fusion protein comprisingInterleukin-10 (“IL-10”) in combination with other inflammatory andimmune regulating cytokines, methods of treating inflammatory and immunedisease or conditions, and/or methods of treating cancer.

INTRODUCTION

IL-10, originally named cytokine synthesis inhibitory factor (Malefyt,Interleukin 10 inhbits cytokine synthesis by human monocytes: Anautoreglatory role of IL-10 produced by monocytes, 1991), is apleiotropic cytokine known to both suppress inflammatory response(Fedorak, 2000), and more recently activate CD8* T cells to induceInterferon γ (“IFNγ”) dependent anti-tumor immune responses (Mumm J. ,2011). IL-10 is a non-covalent homo-dimeric cytokine with structuralsimilarities to IFNγ. IL-10 binds to the IL-10 receptor, which consistsof two subunits of the IL10 receptor 1 (I10R1) and two subunits of theIL-10 receptor 2 (IL10R2) (Moore, 2001). The IL-10 receptor complex isexpressed on the surface of most hematopoietic cells and most highlyexpressed on macrophages and T-cells. While IL-10 has been reported tobe both an immunosuppressive (Schreiber, 2000) and an immunostimulatorycytokine (Mumm, 2011), clinical evaluation of IL-10 treatment of Crohn'spatients resulted in an inverse dose response (Fedorak, 2000; Schreiber,2000), whereas treatment of cancer patients with PEGylated IL-10resulted in dose titratable potent anti-tumor responses (Naing, 2018).PEGylated IL-10 anti-tumor response requires endogenous CD8+T cells andIFNγ (Mumm, 2011). Treatment of tumor bearing animals with PEGylatedIL-10 results in increased intratumor CD8+T cells and increased IFNγ ona per cell basis. Most recently, however, cancer patients treated withPEGylated IL-10 lead to evidence of immune stimulation, but no increasein anti-tumor responses (Spigel, 2020).

Interleukin-2 (“IL-2”) is a four-helix bundle pleiotropic cytokine knownto induce anti-tumor immune responses (Jiang, 2016), but also exhibitinghigh toxicity due to uncontrolled activation of and secretion of IFNγ bynatural killer (“NK”) cells and CD4* T cells and expansion of Tregulatory cells (Chinen, 2016). For this reason, many groups haveattempted to mutate IL-2 to reduce its binding to the high affinityreceptor, in an effort to reduce the toxicity of IL-2 (Chen, 2018).These muteins have not generated substantial clinical success(Bentebibe, 2019). This suggests other mechanisms must be employed toreduce the potentially lethal toxicity of IL-2.

IL-10 has been reported to suppress IL-2 driven IFNγ production secretedby both NK and CD4* T cells (Scott, 2006), but it has also been reportedto act as a cofactor for IL-2 induced CD8* T cell proliferation (Groux,1998). It is therefore not known whether IL-2 and IL-10 will co-activatecells of the immune system or cancel each other out.

Interleukin-4 (“IL-4”) is a four-helix bundle pleiotropic cytokineconsidered the quintessential Th2 driving cytokine (McGuirk, 2000), thatis mostly associated with driving alternative activation by macrophages(Balce, 2011). IL-4 is predominantly associated with drivinginflammation associated with allergic responses and asthma (Steinke,2001; Ryan, 1997). Furthermore, cancer patients have been treated safelywith IL-4 (Davis, 2009), due to IL-4's ability to suppress some cancercell proliferation (Lee, 2016; Gooch, 1998). While IL-4 has beenreported to suppress monocyte secretion of proinflammatory cytokines(Woodward, 2012), it is not considered a potent anti-inflammatorycytokine due to its ability to prime antigen presenting cells and driveproinflammatory cytokine secretion by monocytes exposed to bacteria(Varin, 2010).

It was surprisingly discovered that Epstein-Barr virus (“EBV”) IL-10variants with one or more amino acid substitutions (at amino acidposition 31, 75, or both of the mature EBV IL-10 amino acid sequence ofSEQ ID No. 3) in key IL-10 receptor binding domain regions, altered theability of EBV IL-10 to bind to and activate the IL-10 receptor. Thesemodifications included the ability to increase the affinity of EBV IL-10for the IL-10 receptor. The inventor discovered that EBV IL-10 variantmolecules act as IL-10 receptor agonists capable of treating immunediseases, inflammatory diseases or conditions, and in treating cancer.The inventor also discovered that by incorporating monomeric EBV IL-10variants into a scaffolding system comprising non-immunogenic variableheavy (“VH”) and variable light (“VL”) regions, the resulting EBV IL-10variant molecules were half-life extended, properly folded andfunctionally active. The EBV IL-10 variants incorporated into thescaffolding system showed enhanced IL-10 function on both inflammatorycells (e.g., monocytes/macrophages/dendritic cells) and immune cells(e.g., CD8* T-cells). See, U.S. Pat. No. 10,858,412; filed on Mar. 6,2020 as U.S. application Ser. No. 16/811,718, incorporated by referencein its entirety. This application focuses on a modification to thepreviously described EBV IL-10 scaffolding system to deliver both IL-10and another cytokine as part of a new fusion protein structure thatadditively or synergistically enhances IL-10 biology to treatinflammatory diseases, immune diseases, and/or cancer.

Summary of Various Aspects of the Invention

The present disclosure generally relates to a dual cytokine fusionprotein.

Thus in a first aspect, the present disclosure relates to a dualcytokine fusion protein comprising IL-10 or IL-10 variants as the firstcytokine that is fused to an antigen binding fragment or variable heavy(“VH”) and variable light (“VL”) regions of a monoclonal antibody, and asecond cytokine, wherein the second cytokine is linked in between the VHand VL regions of the antigen binding fragment. In certain embodiments,the first cytokine is an IL-10, such as but not limited to human, mouse,cytomegalovirus, (“CMV”), or EBV IL-10 forms or IL-10 variant molecule,wherein the IL-10 variant has one or more amino acid substitution(s)that impact the IL-10 receptor binding domains. The fusion protein alsoincludes a second cytokine, which is a cytokine that is different fromthe first cytokine, that works in tandem with the IL-10 or IL-10 variantmolecule such that there is an additive or synergistic effect when thefirst and second cytokines are targeted to a specific antigen by thefusion protein or half-life extended by the VH and VL regions of theantigen binding fragment. The fusion protein also includes an antibody,antibody fragment, or antigen binding portion comprising a VH and VLregion that directs the dual cytokine fusion protein to a target antigenrecognized by the VH and VL region of the antibody, antibody fragment,or antigen binding portion thereof. In certain embodiments, the antigenbinding fragment is a scFv.

In yet another aspect, the present disclosure relates to a dual cytokinefusion protein of formula (I):

NH₂—(IL10)—(X¹)—(Z_(n))—(X²)—(IL10)—COOH;

wherein

“IL10” is a monomer of IL-10, wherein the IL-10 is human, mouse, CMV, orEBV IL-10, or a variant thereof, more preferably a IL10 is monomercomprising a sequence selected from SEQ ID Nos: 1, 3, 9, 10, 11, 12, 14,or 16;

“X¹” is a VL or VH region obtained from a first monoclonal antibody;“X²” is a VH or VL region obtained from the first monoclonal antibody;wherein when X¹ is a VL, X² is a VH or when X¹ is a VH, X² is a VL;

“Z is a cytokine other than IL-10; and

“n” is an integer selected from 0-2.

-   -   In yet another aspect, the present disclosure relates to an        IL-10 fusion protein of formula (II)

NH₂—(IL10)-(L)—(X¹)-(L)—(Z_(n))-(L)—(X²)-(L)—(IL10)—COOH;

wherein

“IL-10” is a monomer sequence selected from SEQ ID Nos: 1, 3, 9, 10, 11,12, 14, or 16;

“L” is any linker, more preferably the linker is selected from SEQ IDNo: 39, 40, or 41;

X¹” is a VL or VH region obtained from a first monoclonal antibody; “X²”is a VH or VL region obtained from the first monoclonal antibody;wherein when X¹ is a VL, X² is a VH or when X¹ is a VH, X² is a VL;

“Z” is a cytokine selected from IL-6, IL-4, IL-1, IL-2, IL-3, IL-5,IL-7, IL-8, IL-9, IL-15, IL-21 IL-26, IL-27, IL-28, IL-29, GM-CSF,G-CSF, interferons-α, -β, -γ, TGF-β, or tumor necrosis factors -α, -β,basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13; and

“n” is an integer selected from 0-2.

In other aspects, the present disclosure relates to nucleic acidmolecule that encodes the dual cytokine fusion protein.

In other aspects, the present disclosure relates to methods of makingand purifying the dual cytokine fusion protein. In one embodiment, themethod of making the dual cytokine fusion protein includes recombinantlyexpressing the nucleic acid encoding the dual cytokine fusion protein.

In other aspects, the present disclosure relates to a method of treatingcancer comprising administering to a subject in need thereof, aneffective amount of the dual cytokine fusion protein.

In other aspects, the present disclosure relates to a method of treatinginflammatory diseases or conditions comprising administering to asubject in need thereof, an effective amount of the dual cytokine fusionprotein. Preferably, the inflammatory disease is Crohn's disease,psoriasis, and/or rheumatoid arthritis.

In other aspects, the present disclosure relates to a method of treatingimmune diseases or conditions comprising administering to a subject inneed thereof, an effective amount of the dual cytokine fusion protein.

In other aspects, the present disclosure relates to method of treating,inhibiting, and/or alleviating sepsis and/or septic shock and associatedsymptoms thereof.

The above simplified summary of representative aspects serves to providea basic understanding of the present disclosure. This summary is not anextensive overview of all contemplated aspects, and is intended toneither identify key or critical elements of all aspects nor delineatethe scope of any or all aspects of the present disclosure. Its solepurpose is to present one or more aspects in a simplified form as aprelude to the more detailed description of the disclosure that follows.To the accomplishment of the foregoing, the one or more aspects of thepresent disclosure include the features described and exemplarilypointed out in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a IL-10 cytokine fusion proteindescribed in U.S. Pat. No. 10,858,412.

FIG. 2 is a schematic diagram of a dual cytokine fusion protein embodiedin the present disclosure, wherein the dual cytokine fusion proteincomprises terminally linked IL-10 monomers (or IL-10 variants), where asecond cytokine is incorporated into the linker between the VH and VL ofa scFv.

FIG. 3 is a schematic diagram of a fusion protein comprising twocytokines in an alternate form (termed “SLP-IL-2”) comprising DV07 (ahigh IL-10 receptor affinity variant of EBV IL-10) linked to a VH and VLof a scFv and an IL-2, wherein the IL-2 is fused to the carboxy terminusof the most C-terminal IL-10 monomer.

FIG. 4 is a titration study comparing SLP-IL-2 to IL-10, IL-2, and acombination of IL-10 and IL-2 on the percent reduction of TNFα secretionfrom monocytes/macrophages.

FIG. 5 is a titration study comparing DK2¹⁰ to IL-10 and DegfrDV07 (SLPvariant 3; SEQ ID No: 31) on the percent reduction of TNFα secretionfrom monocytes.

FIG. 6 is a T-cell IFNγ potentiation assay comparing SLP and DK2¹⁰. Thedark gray bar denotes serum trough therapeutic concentrations of bothcytokines, and the light gray bar denotes expected therapeuticconcentration requirements for DK2¹⁰.

FIG. 7 is an assay to determine the effects of IL-10 on NK cells, CD4*T-cells, and CD8* T-cells on IL-2 mediated induction of IFNγ. The darkgray bar denotes serum trough therapeutic concentrations of bothcytokines, and the light gray bar denotes expected therapeuticconcentration requirements for DK2¹⁰.

FIG. 8 is an assay measuring the effects of cytokines on model antigenpresentation in T cells.

FIG. 9 is an assay measuring the induction of IFNγ in CD4* and CD8* Tcells after antigen exposure.

FIG. 10 is an in vivo CT26 (hEGFR*) tumor mouse model study comparinganti-tumor effects in mice treated with Degfr:DV07 or DK2¹⁰.

FIG. 11 is an in vivo CT26 (hEGFR*) tumor mouse model study comparingthe weight of mice treated with Degfr:DV07 or DK2¹⁰.

FIG. 12 is an in vivo CT26 (hEGFR*) tumor mouse model study comparingsurvival of mice treated Degfr:DV07 and DK2¹⁰.

FIG. 13 is a titration study for IL-10, IL-4, and IL-10 and IL-4 on thepercent reduction of TNFα secretion from monocytes.

FIG. 14 is a titration study for IL-10, IL-4, IL-4 and DeboWtEBV, andDeboWtEBV alone on the percent reduction of TNFα secretion frommonocytes.

FIG. 15 is a T-cell IFNγ potentiation assay comparing DeboWtEBV and IL-4against DeboWtEBV alone.

FIG. 16 is a titration study evaluating of IL-10, IL-4, DeboDV06, andDeboDV06 in combination with IL-4 on suppressing LPS induced TNFαsecretion by monocytes/macrophages.

FIG. 17 is a schematic representation of the class of moleculesdesignated as the DK4¹⁰ form.

FIG. 18 is a titration study evaluating IL-4DeboDV06 in DK4¹⁰ form (alsoknown as “4DeboDV06”) in comparison to IL-10, IL-4, DeboDV06, and IL-10in combination with IL-4 on suppressing LPS induced TNFα secretion bymonocytes/macrophages.

FIG. 19 is a titration study evaluating IL-4DeboDV06 in DK4¹⁰ form (alsoknown as “4DeboDV06”) in comparison to IL-10, IL-4, DeboDV06, andDeboDV06 in combination with IL-4 on CD8+T cells.

FIG. 20 is a titration study evaluating IL-4HADeglymCD14DV06 andIL-4HADeglymCD14DV07, which are members of the DK4¹⁰ class of moleculescomprising a non-glycosylated (N38A) and high affinity (T13D) form ofhuman IL-4, and compared to IL-10, IL-4, and IL-4DeboDV06 (also known as“4DeboDV06”) in DK4¹⁰ form on suppressing LPS induced TNFα secretion bymacrophage/monocytes.

FIG. 21 is a titration study evaluating IL-4ngDmCD14DV06 andIL-4ngDmCD14DV07, which are members of the DK4¹⁰ class of moleculescomprising a single substitution at N38A resulting in a non-glycosylatedform of IL4, and compared to IL-10 on suppressing LPS induced TNFαsecretion by monocytes/macrophages.

FIG. 22 is a titration study evaluating IL-4ngDmCD14DV06 andIL-4ngDmCD14DV07, which are members of the DK4¹⁰ class of moleculescomprising a single substitution at N38A resulting in a non-glycosylatedform of IL4, and compared to IL-10 on mediating IFNγ induction by CD8+Tcells.

FIG. 23 is a titration study evaluating IL-4ngDmMAdCAMDV06, which aremembers of the DK4¹⁰ class of molecules comprising a single substitutionat N38A resulting in a non-glycosylated form of IL4, and compared toIL-10 on suppressing LPS induced TNFα secretion by monocytes/macrophage.

FIG. 24 is a titration study evaluating IL-4ngDmMAdCAMDV06, which aremembers of the DK4¹⁰ class of molecules comprising a single substitutionat N38A resulting in a non-glycosylated form of IL4, and compared toIL-10 on mediating IFNγ induction by CD8+T cells.

FIG. 25 is an in vivo sepsis mouse model study comparing survival ofmice treated with IL-4ngDmMAdCAMDV06 before and after LPSadministration.

DETAILED DESCRIPTION

Exemplary aspects are described herein in the context of a dual cytokinefusion protein comprising IL-10, methods of making the dual cytokinefusion protein comprising IL-10, and methods of using the dual cytokinefusion protein comprising IL-10 for treating inflammatory diseases orconditions, immune diseases or conditions, treating and/or preventingcancer. Those of ordinary skill in the art will realize that thefollowing description is illustrative only and is not intended to be inany way limiting. Other aspects will readily suggest themselves to thoseskilled in the art having the benefit of this disclosure. Reference willnow be made in detail to implementations of the exemplary aspects asillustrated in the accompanying drawings. The same reference indicatorswill be used to the extent possible throughout the drawings and thefollowing description to refer to the same or like items.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the variousdescribed embodiments, the preferred materials and methods are describedherein.

Unless otherwise indicated, the embodiments described herein employconventional methods and techniques of molecular biology, biochemistry,pharmacology, chemistry, and immunology, well known to a person skilledin the art. Many of the general techniques for designing and fabricatingthe IL-10 variants, including but not limited to human, mouse, CMVand/or EBV forms of IL-10, as well as the assays for testing the IL-10variants, are well known methods that are readily available and detailedin the art. See, e.g., Sambrook, et al., Molecular Cloning: A LaboratoryManual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N.Kaplan eds., Academic Press, Inc.); Handbook of Experimental Immunology,Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell ScientificPublications); A. L. Lehninger, Biochemistry (Worth Publishers, Inc.,current addition). N-terminal aldehyde based PEGylation chemistry isalso well known in the art.

Definitions

The following terms will be used to describe the various embodimentsdiscussed herein, and are intended to be defined as indicated below.

As used herein in describing the various embodiments, the singular forms“a”, “an” and “the” include plural referents unless the content clearlydictates otherwise.

The term “about”, refers to a deviance of between 0.0001-5% from theindicated number or range of numbers. In one embodiment, the term“about”, refers to a deviance of between 1-10% from the indicated numberor range of numbers. In one embodiment, the term “about”, refers to adeviance of up to 25% from the indicated number or range of numbers. Ina more specific embodiment, the term “about” refers to a difference of1-25% in terms of nucleotide sequence homology or amino acid sequencehomology when compared to a wild-type sequence.

The term “interleukin-10” or “IL-10” refers to a protein comprising twosubunits non-covalently joined to form a homodimer, where IL-10 is anintercalated dimer of two six helix bundle (helix A-F). As used herein,unless otherwise indicated “interleukin-10” and “IL-10” refers to anyform of IL-10, including but not limited to human IL-10 (“hlL-10”;Genbank Accession No. NP_000563; or U.S. Pat. No. 6,217,857) protein(SEQ ID No: 1) or nucleic acid (SEQ ID No: 2); mouse IL-10 (“mIL-10”;Genbank Accession No: M37897; or U.S. Pat. No. 6,217,857) protein (SEQID No: 7) or nucleic acid (SEQ ID No: 8); or viral IL-10, (“vIL-10”).Viral IL-10 homologs may be derived from EBV or CMV (Genbank AccessionNos. NC_007605 and DQ367962, respectively). The term EBV-IL10 refers tothe EBV homolog of IL-10 protein (SEQ ID No: 3) or the nucleic acid (SEQID No: 4). The term CMV-IL10 refers to the CMV homolog of IL-10 protein(SEQ ID No: 5) or the nucleic acid (SEQ ID No: 6). The term “monomeric”or “monomer of” IL-10, as used herein, refers to the individual subunitsof IL-10 or variant IL-10 that, when non-covalently joined, form ahomodimer of IL-10 or variant IL-10. The terms “wild-type,” “wt” and“native” are used interchangeably herein to refer to the sequence of theprotein (e.g. IL-10, CMV-IL10 or EBV IL-10) as commonly found in naturein the species of origin of the specific IL-10 in question. For example,the term “wild-type” or “native” EBV IL-10 would thus correspond to anamino acid sequence that is most commonly found in nature.

The terms “variant,” “analog” and “mutein” refer to biologically activederivatives of the reference molecule, that retain a desired activity,such as, for example, anti-inflammatory activity. Generally, the terms“variant,” “variants,” “analog” and “mutein” as it relates to apolypeptide refers to a compound or compounds having a nativepolypeptide sequence and structure with one or more amino acidadditions, substitutions (which may be conservative in nature), and/ordeletions, relative to the native molecule. As such, the terms “IL-10variant”, “variant IL-10,” “IL-10 variant molecule,” and grammaticalvariations and plural forms thereof are all intended to be equivalentterms that refer to an IL-10 amino acid (or nucleic acid) sequence thatdiffers from wild-type IL-10 anywhere from 1-25% in sequence identity orhomology. Thus, for example, an EBV IL-10 variant molecule is one thatdiffers from wild-type EBV IL-10 by having one or more amino acid (ornucleotide sequence encoding the amino acid) additions, substitutionsand/or deletions. Thus in one form, an EBV IL-10 variant is one thatdiffers from the wild type sequence of SEQ ID No.:3 by having about 1%to 25% difference in sequence homology, which amounts to about 1-42amino acid difference. In one embodiment, an IL-10 variant is an EBVIL-10 comprising a V31L amino acid mutation (“DV05”; SEQ ID No: 12), aA751 amino acid mutation (“DV06”; SEQ ID No: 14), or both V31L and aA751 amino acid mutations (“DV07”; SEQ ID No: 16).

The term “fusion protein” refers to a combination or conjugation of twoor more proteins or polypeptides that results in a novel arrangement ofproteins that do not normally exist naturally. The fusion protein is aresult of covalent linkages of the two or more proteins or polypeptides.The two or more proteins that make up the fusion protein may be arrangedin any configuration from amino-terminal end (“NH₂”) to carboxy-terminalend (“COOH”). Thus for example, the carboxy-terminal end of one proteinmay be covalently linked to either the carboxy terminal end or the aminoterminal end of another protein. Exemplary fusion proteins may includecombining a monomeric IL-10 or a monomeric variant IL-10 molecule withone or more antibody variable domains (i.e., VH and/or VL) or singlechain variable region (“scFv”). The fusion proteins may also form dimersor associated with other fusion proteins of the same type, which resultsin a fusion protein complex. The complexing of the fusion protein may insome cases activate or increase the functionality of a fusion proteinwhen compared to a non-complexed fusion protein. For example, amonomeric IL-10 or monomeric variant IL-10 molecule with one or moreantibody variable domains may have limited or decreased capacity to bindto an IL-10 receptor; however, when the fusion protein is complexed, themonomeric forms of IL-10 or variant IL-10 molecule become a homodimerand the variable domains associate into a functional diabody.

The term “homolog,” “homology,” “homologous” or “substantiallyhomologous” refers to the percent identity between at least twopolynucleotide sequences or at least two polypeptide sequences.Sequences are homologous to each other when the sequences exhibit atleast about 50%, preferably at least about 75%, more preferably at leastabout 80%-85%, preferably at least about 90%, and most preferably atleast about 95%-98% sequence identity over a defined length of themolecules.

The term “sequence identity” refers to an exact nucleotide-by-nucleotideor amino acid-by-amino acid correspondence. The sequence identity mayrange from 100% sequence identity to 50% sequence identity. A percentsequence identity can be determined using a variety of methods includingbut not limited to a direct comparison of the sequence informationbetween two molecules (the reference sequence and a sequence withunknown percent identity to the reference sequence) by aligning thesequences, counting the exact number of matches between the two alignedsequences, dividing by the length of the reference sequence, andmultiplying the result by 100. Readily available computer programs canbe used to aid in the identification of percent identity.

The terms “subject,” “individual” or “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, murine, rodent, simian, human, farm animals,sport animals, and certain pets.

The term “administering” includes routes of administration which allowthe active ingredient of the application to perform their intendedfunction.

A “therapeutically effective amount” as it relates to, for example,administering the EBV IL-10 variants or fusion proteins thereofdescribed herein, refers to a sufficient amount of the EBV IL-10 variantor fusion proteins thereof to promote certain biological activities.These might include, for example, suppression of myeloid cell function,enhanced Kupffer cell activity, and/or lack of any effect on CD8* Tcells or enhanced CD8* T-cell activity as well as blockade of mast cellupregulation of Fc receptor or prevention of degranulation. Thus, an“effective amount” will ameliorate or prevent a symptom or sign of themedical condition. Effective amount also means an amount sufficient toallow or facilitate diagnosis.

The term “treat” or “treatment” refers to a method of reducing theeffects of a disease or condition. Treatment can also refer to a methodof reducing the underlying cause of the disease or condition itselfrather than just the symptoms. The treatment can be any reduction fromnative levels and can be, but is not limited to, the complete ablationof the disease, condition, or the symptoms of the disease or condition.

The following table provides definitions for the various IL-10 fusionproteins and dual cytokine fusions proteins comprising IL-10 referencedin the present disclosure:

Term Definition “Debo” Refers to the base half-life extended IL-10scaffolding system schematically represented by FIG. 1, wherein monomersof IL-10 (e.g., SEQ ID No. 1, 3, or 5) or IL-10 variant molecules (e.g.SEQ ID No: 9-11, 12, 14, or 16) are linked to a scFv comprising VH andVL regions obtained from a human anti-ebola antibody. Without beingbound to any particular theory, the scaffolding system is capable offorming a stable complex due to VH and VL pair formation and thehomodimerization of the IL- 10 monomers. “DeboWtEBV” or Refers to Deboschematically represented by FIG. 1, the “DeboWt” molecule comprisingmonomers of wild type EBV IL-10 (SEQ ID No: 3) linked to a scFvcomprising VH and VL regions obtained from a human anti-ebola antibody.“DeboDV06” Refers to Debo schematically represented by FIG. 1, themolecule comprising monomers of IL-10 variant DV06 (SEQ ID No: 14)linked to a scFv comprising VH and VL regions obtained from a humananti-ebola antibody. “DeboDV07” Refers to Debo schematically representedby FIG. 1, the molecule comprising monomers of IL-10 variant DV07 (SEQID No: 16) linked to a scFv comprising VH and VL regions obtained from ahuman anti-ebola antibody. “DegfrDV07” Refers to a Debo schematicallyrepresented by FIG. 1, the molecule comprising monomers of IL-10 variantDV07 and where the 3 CDRs in the VH and the 3 CDRs in the VL regionsfrom the human anti-ebola scFv are replaced by 3 CDRs in the VH and 3CDRs in the VL from an anti- EGFR antibody (Cetuximab). “SLP” Refers toan optimized variant form (variant #3) of DegfrDV07 that is SEQ ID No:31. “IL4DeboDV06” or Refers to a dual cytokine fusion proteinschematically “4DeboDV06” or represented by FIG. 17, where DeboDV06includes a “DK4¹⁰DV06” wild-type human IL-4 (SEQ ID No: 43) linkedbetween the human anti-ebola derived scFv region. “IL4DeboDV07” orRefers to a dual cytokine fusion protein schematically “4DeboDV07” orrepresented by FIG. 2, where DeboDV07 includes a wild “DK4¹⁰DV07” typehuman IL-4 (SEQ ID No: 43) linked between the human anti-ebola derivedscFv region. “DK2¹⁰” or Refers to a class of dual cytokine fusionprotein “DK2¹⁰ form” molecules schematically represented by FIG. 2, themolecule where DeboDV07 includes a human IL-2 (SEQ ID No: 36) linkedbetween the human anti-ebola derived scFv region. DK2¹⁰ may be made intoa targeting molecule by optionally replacing the 6 CDR regions from thehuman anti-ebola derived scFv with 6 CDR regions (3 CDRs in the VH and 3CDRs in the VL) from any monoclonal antibody. The nomenclature willfollow the format of “DK2¹⁰(protein target)”. For example, if DK2¹⁰includes engraftment of 6 CDRs from a human anti-EGFR antibody(cetuximab), the molecule will be termed DK2¹⁰egfr (SEQ ID No: 35) or ifDK2¹⁰ includes engraftment of the 6 CDRs from a human anti-HER2/Neuantibody (trastuzumab), the molecule will be termed DK2¹⁰her2 (SEQ IDNo: 52-54, or 55), respectively; or if DK2¹⁰ includes engraftment of 6CDRs from a human anti-VEGFR1 or anti-VEGFR2 antibody, the molecule willbe termed DK2¹⁰vegfr1 or DK2¹⁰vegfr2, respectively; or if DK2¹⁰ includesengraftment of 6 CDRs from a human anti-PDGFR antibody, the moleculewill be termed DK2¹⁰pdgfr. “DK2¹⁰egfr” Refers to a DK2¹⁰ moleculetargeting EGFR, where the 6 CDR regions from the human anti-eboladerived scFv region are replaced by the 6 CDR regions (3 CDRs in the VHand 3 CDRs in the VL) from a human anti-EGFR antibody (cetuximab). Themolecule is SEQ ID No: 35. The molecule may also include optimized VH(SEQ ID No: 37) and VL (SEQ ID No: 38) regions. “DK2¹⁰her2” Refers to aDK2¹⁰ molecule targeting HER2, where the 6 CDR regions from the humananti-ebola derived scFv region are replaced by the 6 CDR regions (3 CDRsin the VH and 3 CDRs in the VL) from a human anti-HER2 antibody(trastuzumab). The molecule is SEQ ID No: 52- 54, or 55. “DK2¹⁰vegfr1”Refers to a DK2¹⁰ molecule targeting VEGFR1, where the 6 CDR regionsfrom the human anti-ebola derived scFv region are replaced by the 6 CDRregions (3 CDRs in the VH and 3 CDRs in the VL) from a human anti-VEGFR1antibody. “DK2¹⁰vegfr2” Refers to a DK2¹⁰ molecule targeting VEGFR2,where the 6 CDR regions from the human anti-ebola derived scFv regionare replaced by the 6 CDR regions (3 CDRs in the VH and 3 CDRs in theVL) from a human anti-VEGFR2 antibody. “DK4¹⁰” or Refers to a class ofdual cytokine fusion protein “DK4¹⁰ form” molecules schematicallyrepresented by FIG. 2 or FIG. 17, the molecule comprising eitherDeboDV06 or DeboDV07 in combination with an IL-4 (SEQ ID No: 43) or IL-variants (SEQ ID No: 44 or 45) where the IL-4 or IL- 4 variant is linkedin the hinge region of a human anti- ebola derived scFv region. DK4¹⁰may be made into a targeting molecule by optionally replacing the 6 CDRregions from the human anti-ebola derived scFv with 6 CDR regions (3CDRs in the VH and 3 CDRs in the VL) from any monoclonal antibody. Forexample, if DK4¹⁰ includes engraftment of 6 CDRs from a mouse anti-CD14antibody in combination with DV06 or DV07, the molecule will be termedDK4¹⁰mCD14DV06 (SEQ ID No: 49) or DK4¹⁰mCD14DV07 (SEQ ID No: 50),respectively; or if DK4¹⁰ includes engraftment of 6 CDRs from a mouseanti-MAdCAM antibody in combination with DV06, the molecule will betermed DK4¹⁰mMAdCAMDV06 or DK4¹⁰mMAdCAM (SEQ ID No: 51); or if DK4¹⁰includes engraftment of 6 CDRs from a human anti-VEGFR1 or humananti-VEGFR2 antibody, the molecule will be termed DK4¹⁰vegfr1 orDK4¹⁰vegfr2, respectively, where the IL-4 moiety is the non-glycosylatedform of IL-4 (a N38A IL-4 variant of SEQ ID Nos: 44) and DV06.“DK4¹⁰ngDV06mCD14” or Refers to a DK4¹⁰ molecule (schematicallyrepresented “DK4¹⁰mCD14DV06” by FIG. 17) targeting mouse CD14, themolecule comprising DeboDV06 with an non-glycosylated form of IL-4 (aN38A IL-4 variant of SEQ ID Nos: 44) linked in the hinge region of thehuman anti-ebola derived scFv region. The 6 CDR regions from the humananti-ebola derived scFv are replaced by the 6 CDR regions (3 CDRs in theVH and 3 CDRs in the VL) from a mouse anti-CD14 antibody. This moleculeis SEQ ID No: 49. “DK4¹⁰ngDV07mCD14” or Refers to a DK4¹⁰ molecule(schematically represented “DK4¹⁰mCD14DV07” by FIG. 1) targeting mouseCD14, the molecule comprising DeboDV07 with a non-glycosylated form ofIL- 4 (a N38A IL-4 variant of SEQ ID Nos: 44) linked in the hinge regionof the human anti-ebola derived scFv region. The 6 CDR regions from thehuman anti-ebola derived scFv are replaced by the 6 CDR regions (3 CDRsin the VH and 3 CDRs in the VL) from a mouse anti-CD14 antibody. Themolecule is SEQ ID No: 50. “DK4¹⁰ngDV06mMAdCAM” or Refers to a DK4¹⁰molecule (schematically represented “DK4¹⁰mMAdCAMDV06” or by FIG. 17)targeting mouse MAdCAM, the molecule “DK4¹⁰mMAdCAM” comprising DeboDV06with a non-glycosylated form of IL- 4 (a N38A IL-4 variant of SEQ IDNos: 44) linked in the hinge region of the human anti-ebola derived scFvregion. The 6 CDR regions from the human anti-ebola derived scFv arereplaced by the 6 CDR regions (3 CDRs in the VH and 3 CDRs in the VL)from a mouse anti-CD14 antibody. The molecule is SEQ ID No: 51.“DK4¹⁰ngDV06CD14” or Refers to a DK4¹⁰ molecule (schematicallyrepresented “DK4¹⁰CD14DV06” by FIG. 17) targeting human CD14, themolecule comprising DeboDV06 with an non-glycosylated form of IL-4 (aN38A IL-4 variant of SEQ ID Nos: 44) linked in the hinge region of thehuman anti-ebola derived scFv region. The 6 CDR regions from the humananti-ebola derived scFv are replaced by the 6 CDR regions (3 CDRs in theVH and 3 CDRs in the VL) from a human anti-CD14 antibody. This moleculeis SEQ ID No: 56-58, or 59. “DK4¹⁰ngDV06vegfr1” or Refers to a DK4¹⁰molecule (schematically represented “DK4¹⁰vegfr1 DV06” by FIG. 17)targeting human VEGFR1, the molecule comprising DeboDV06 with annon-glycosylated form of IL-4 (a N38A IL-4 variant of SEQ ID Nos: 44)linked in the hinge region of the human anti-ebola derived scFv region.The 6 CDR regions from the human anti-ebola derived scFv are replaced bythe 6 CDR regions (3 CDRs in the VH and 3 CDRs in the VL) from a humananti-VEGFR1 antibody. “DK4¹⁰ngDV06vegfr2” or Refers to a DK4¹⁰ molecule(schematically represented “DK4¹⁰vegfr2DV06” by FIG. 17) targeting humanVEGFR2, the molecule comprising DeboDV06 with an non-glycosylated formof IL-4 (a N38A IL-4 variant of SEQ ID Nos: 44) linked in the hingeregion of the human anti-ebola derived scFv region. The 6 CDR regionsfrom the human anti-ebola derived scFv are replaced by the 6 CDR regions(3 CDRs in the VH and 3 CDRs in the VL) from a human anti-VEGFR2antibody.

Dual Cytokine Fusion Protein Structure

The present disclosure provides an improvement on an embodiment of anIL-10 fusion protein previously described in U.S. Pat. No. 10,858,412(filed as U.S. application Ser. No. 16/811,718), which is incorporatedby reference in its entirety. The improvement to the IL-10 fusionprotein includes incorporating a second cytokine molecule into thepreviously described IL-10 fusion protein. FIG. 1 is a schematic diagramrepresenting one of the previously disclosed IL-10 fusion proteinconstructs described in U.S. Pat. No. 10,858,412. This IL-10 fusionprotein is constructed on a VH and VL scFv scaffolding featuring twomonomers of IL-10 on each end (i.e., a first IL-10 monomer on the aminoterminal end and a second IL-10 monomer on the carboxy terminal end).The primary scaffolding system comprises a scFv obtained from a humananti-ebola antibody. The IL-10 fusion protein described in U.S. Pat. No.10,858,412 includes 6 complementarity-determining regions (“CDRs”)having CDRs 1-3 in the VH and CDRs 1-3 in the VL. Optionally, the VH andVL regions are capable of targeting the IL-10 fusion protein to aspecific antigen. This is accomplished by substituting the 6 CDR regionsof the VH and VL pair (3 CDRs in the VH and 3 CDRs in the VL) with 6 CDRregions from a VH and VL of a receptor or antigen targeting antibody, orantigen binding fragment thereof. The ability to substitute and optimizethe 6 CDR and framework regions and to engraft these CDRs into the scFvscaffolding described herein, is well known and practiced by those ofskill in the art. These 6 CDR regions are substitutable with 6 CDRs fromany monoclonal antibody, which any person of skill would be capable ofdetermining based on the specific target of interest.

In a first aspect, the present application relates to a dual cytokinefusion protein comprising IL-10 and at least one other cytokine, wherebythe dual cytokine fusion protein has a combined or synergisticfunctionality when compared to the IL-10 fusion protein previouslydescribed in U.S. Pat. No. 10,858,412. FIG. 2 is a representativediagram of the improved dual cytokine fusion protein comprising IL-10.In particular, the improved dual cytokine fusion protein adapts the sameor substantially same scaffolding system made up of a VH and VL scFvwhereby two monomers of IL-10 terminate the dual fusion protein at theamino and carboxy terminal ends. The second cytokine is conjugated tothe IL-10 fusion protein by being fused between the VH and VL regions ofthe scFv, which is the hinge region of the scFv. The dual cytokinefusion protein is capable of forming a functional protein complexwhereby the monomers of IL-10 homodimerize into a functional IL-10molecule and the VH and VL regions form a pair that associate togetherto form a scFv complex that permits antigen binding and recognition.

In certain embodiments, the dual cytokine fusion protein comprisingIL-10 is a structure having formula I

NH₂—(IL10)—(X¹)—(Z_(n))—(X²)—(IL10)—COOH

wherein

-   -   “IL-10” is any IL-10 monomer, such as but not limited to human,        mouse, CMV or EBV IL-10, or IL-10 variant molecules;    -   “X¹” is a VL or VH region obtained from a first monoclonal        antibody;    -   “X²” is a VH or VL region obtained from the first monoclonal        antibody,    -   wherein when X¹ is a VL, X² is a VH or when X¹ is a VH, X² is a        VL;    -   “Z” is a second cytokine, wherein the second cytokine is a        cytokine other than IL-10; and    -   “n” is an integer selected from 0-2.

In another embodiment, the dual cytokine fusion protein comprising IL-10is a structure having formula II

NH₂—(IL10)-(L)—(X¹)-(L)—(Z_(n))-(L)—(X²)-(L)—(IL10)—COOH

wherein

-   -   “IL-10” is an IL-10 monomer;    -   “L” is a linker, preferably a linker of SEQ ID NO.: 39, 40, or        41;    -   “X¹” is a VL or VH region obtained from a first monoclonal        antibody;    -   “X²” is a VH or VL region obtained from the first monoclonal        antibody;    -   wherein when X¹ is a VL, X² is a VH or when X¹ is a VH, X² is a        VL;    -   “Z” is a second cytokine; and    -   “n” is an integer selected from 0-2.

In one embodiment, the IL-10 monomer includes any form of IL-10including human (SEQ ID NO.:1), CMV (SEQ ID NO.: 5), EBV (SEQ ID NO.:3),or mouse (SEQ ID No: 7). In another embodiment, the IL-10 monomer is amodified or variant form of EBV IL-10 (SEQ ID NO.: 3), including thosethat are described in U.S. Pat. No. 10,858,412. In a preferredembodiment, the EBV IL-10 comprises one or more substitutions in SEQ IDNo. 3 at amino acid position 31 (herein termed “DV05”), 75 (hereintermed “DV06”), or both (herein termed “DV07”). In yet anotherembodiment, the IL-10 monomer is a sequence of SEQ ID No: 9, 10, 11, 12,14, or 16. The first and second monomers of IL-10 or IL-10 variantmolecule are each located at the terminal ends of the fusion protein(i.e., the first monomer at the amino terminal end and the secondmonomer at the carboxy terminal end) as represented by FIG. 1.

In another embodiment, the VH and VL regions are from an antibody,antibody fragment, or antigen binding fragment thereof. The antigenbinding fragment includes, but is not limited to, a scFv, Fab, F(ab′)₂,V-NAR, diabody, or nanobody. Preferably the VH and VL, are from a singlechain variable fragment (“scFv”).

In another embodiment, the dual cytokine fusion protein comprising IL-10includes a VH and VL pair from a single antibody. The VH and VL pair actas a scaffolding onto which monomers of IL-10 or variants thereof may beattached such that the monomers of IL-10 or variants thereof may be ableto homodimerize into a functioning IL-10 molecule. A person of skill inthe art will therefore appreciate that the VH and VL scaffolding used inthe fusion protein may be selected based on the desired physicalattributes needed for proper homodimerization of the IL-10 monomers orIL-10 monomer variants and/or the desire to maintain VH and VL targetingability. Likewise, a person of skill will also understand that the 6CDRs within the VH and VL pair (3 CDRs from the VH and 3 CDRs from VL)may also be substituted with 6 CDRs from other antibodies to obtain aspecifically targeted fusion protein. In one embodiment, 3 CDRs from aVH and 3 CDRs from a VL (i.e., a VH and VL pair) of any monoclonalantibody may be engrafted into a scaffolding system comprising SEQ Nos:18, 20, 21, 23, 24, or 25. It is also envisioned that if the fusionprotein is not intended to target any specific antigen, a VH and VL pairmay be selected as the scaffolding that does not target any particularantigen (or is an antigen in low abundance in vivo), such as the VH andVL pair from an anti-HIV and/or anti-Ebola antibody. Thus, in anembodiment, the IL-10 fusion protein of the present application mayinclude a VH and VL pair from a human anti-ebola antibody, morepreferably a sequence of SEQ ID No: 18, 21, or 25. The fusion proteinmay comprises a range of 1-4 variable regions. In another embodiment,the variable regions may be from the same antibody or from at least twodifferent antibodies.

In another embodiment, the target specificity of the antibody variablechains or VH and VL pair or the 6 CDRs of the VH and VL pair mayinclude, but not limited to those targeting proteins, cellularreceptors, and/or tumor associated antigens. In another embodiment, theCDR regions from any VH and VL pair may be engrafted into thescaffolding system described above, such scaffolding preferably includesa system termed Debo (schematically represented by FIG. 1), wherebyIL-10 monomers are linked to a scFv comprising VH and VL regions of ahuman anti-ebola antibody and the second cytokine is linked in the hingeregion of the scFv (schematically represented by FIG. 2). Morepreferably engraftment into the Debo scaffolding system occurs in ascaffolding comprising a sequence of SEQ ID No: 18, 20, 21, 23, 24, or25. In yet another embodiment, the variable regions or VH and VL pair orthe 6 CDRs of the VH and VL pair are obtained from antibodies thattarget antigens associated with various diseases (e.g., cancer) or thosethat are not typically found or rarely found in the serum of a healthysubject, for example variable regions from antibodies directed to EGFR,PDGFR, VEGFR1, VEGFR2, Her2Neu, FGFR, GPC3, or other tumor associatedantigens, MAdCam, ICAM, VCAM, CD14 or other inflammation associated cellsurface proteins, HIV and/or Ebola. Thus, in one embodiment, thevariable regions are obtained or derived from anti-EGFR, anti-MAdCam,anti-HIV (Chan et al, J. Virol, 2018, 92(18):e00641 1-19), anti-ICAM,anti-VCAM, anti-CD14, or anti-Ebola (US Published Application2018/0180614, incorporated by reference in its entirety, especially mAbsdescribed in Tables 2, 3, and 4) antibodies, for example. In anotherembodiment, the variable regions are obtained or derived from antibodiescapable of enriching the concentration of cytokines, such as IL-10, to aspecific target area so as to enable IL-10 to elicit its biologicaleffect. Such an antibody might include those that target overexpressedor upregulated receptors or antigens in certain diseased regions orthose that are specifically expressed in certain impacted areas. Forexample, the variable regions might be obtained from antibodies specificfor epidermal growth factor receptor (EGFR); CD52; CD14; various immunecheck point targets, such as but not limited to PD-L1, PD-1, TIM3, BTLA,LAG3 or CTLA4; CD20; CD47; GD-2; VEGFR1; VEGFR2; HER2; PDGFR; EpCAM;ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβTrap; MAdCam, β7 integrin subunit; α4β7 integrin; α4 integrin SR-A1;SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2;SR-G; SR-H1; SR-H2; SR-11; and SR-J1 to name a few. A monomer of IL-10(e.g., human, CMV, or EBV) or variant IL-10 molecule (described herein)is conjugated to either the amino terminal end or the carboxy terminalend of a variable region (VH or VL), such that the monomer IL-10 orvariant IL-10 molecule is able to dimerize with one another. In apreferred embodiment, the monomers of IL-10 (or variant IL-10) are fusedto the VH and VL pair in accordance to formula I or II, wherein theIL-10 monomer is an EBV IL-10, DV05, DV06, or DV07 form of IL-10.

The dual cytokine fusion protein or dual cytokine fusion protein complexmay also have an antigen targeting functionality. The dual cytokinefusion protein or dual cytokine fusion protein complex will comprise aVH and VL pair that is able to associate together to form an antigenbinding site or ABS. In some configurations, the IL-10 monomers or IL-10variant monomers thereof will be covalently linked to the end comprisingthe antigen binding site. The variable regions may be further modified(e.g., by addition, subtraction, or substitution) by altering one ormore amino acids that reduce antigenicity in a subject. Othermodifications to the variable region may include amino acidssubstitutions, deletions, or additions that are found outside of the 6CDR regions of the VH and VL regions and serve to increase stability andexpression of the VH and VL regions of the scFv. For example, themodifications may include modifications that are described in SEQ ID No:27, 29, 31, or 33 wherein the CDR regions are obtained from the VH andVL regions of an anti-EGFR antibody and the regions outside of the CDRsare optimized to stabilize the scFv and/or optimized to increaseexpression, which may be used as a basis for linking the second cytokinebetween the VH and VL regions of the scFv. To demonstrate that thesetypes of modifications are within the purview of a skilled artisan,similar modifications to the CDR regions and regions outside of the CDRswere made to a molecule in DK2¹⁰ form comprising DV07 and targetinghuman HER2 (i.e., DK2¹⁰her2), such as those described in SEQ ID No:52-54, or 55, more preferably SEQ ID No: 54 (variant 4) or 55 (variant5). Moreover, modifications to the CDR regions and regions outside ofthe CDRs were made to a molecule in DK4¹⁰ form comprising DV06 andtargeting human CD14 (i.e., DK4¹⁰CD14DV06), such as those described inSEQ ID No: 56-58, or 59, more preferably SEQ ID No: 56 (variant 2).These and other modifications may also be made to a molecule in DK2¹⁰form comprising DV07 and targeting human VEGFR1 or VEGFR2; or to amolecule in DK4¹⁰ form comprising DV06 and targeting human VEGFR1 orVEGFR2. A person of skill in the art would be capable of determiningother modifications that stabilize the scFv and/or to optimize thesequence for purposes of expression.

The VH and VL pair form a scaffolding onto which CDR regions obtainedfor a plurality of antibodies may be grafted or engrafted. Such antibodyCDR regions include those antibodies known and described above. The CDRregions in the above described VH and VL scaffolding will include thefollowing number of amino acid positions available for CDRengraftment/insertion:

Heavy chain CDR1 3-7 amino acids Heavy chain CDR2 7-11 amino acids Heavychain CDR3 7-11 amino acids Light chain CDR1 9-14 amino acids Lightchain CDR2 5-9 amino acids Light chain CDR3 7-11 amino acids

In a preferred embodiment, the dual cytokine fusion protein comprisingIL-10 will include the previously described scaffolding IL-10 fusionprotein where the VH and VL pair is derived from an anti-ebola antibody(such as those described in SEQ ID No: 19, 27, 29, 31, and 33) wherebythe 6 CDR regions from the anti-ebola antibody are removed and engraftedwith a VH and VL pair of a specific targeting antibody, such as but notlimited to EGFR; CD52; CD14; various immune check point targets, such asbut not limited to PD-L1, PD-1, TIM3, BTLA, LAG3 or CTLA4; CD20;CD47;GD-2; VEGFR1; VEGFR2; HER2; PDGFR; EpCAM; ICAM (ICAM −1, −2, −3,−4, −5), VCAM, CD14, FAPα; 5T4; Trop2; EDB-FN; TGFβ Trap; MAdCam, β7integrin subunit; α4β7 integrin; α4 integrin SR-A1; SR-A3; SR-A4; SR-A5;SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2;SR-11; and SR-J1. In an embodiment, the 6 anti-ebola CDR regions aresubstituted with 6 CDR regions from anti-EGFR, anti-MAdCAM, anti-VEGFR1,anti-VEGFR2, anti-PDGFR, or anti-CD14. In a preferred embodiment, theIL-10 fusion protein is a sequence of SEQ ID No: 18, 20, 21, 23, 24, or25 to which any of the CDRs from the above described antibodies may beengrafted. In a more preferred embodiment, the IL-10 fusion protein is asequence of SEQ ID No: 19, 22, or 26. In a preferred embodiment, asecond cytokine, such as but not limited to IL-2, IL-4, IFNα, is linkedin the hinge region between the VH and VL of the scFv obtained from ahuman anti-ebola antibody from an IL-10 fusion protein having a sequenceof SEQ ID No: 18−27, 29, 31, or 33.

In yet another embodiment, the second cytokine, is fused between the VHand VL of a scFv, as depicted in FIG. 2. The second cytokine isconjugated between the VH or VL region such that the second cytokineretains its functional properties. In one embodiment, the secondcytokine is different from the IL-10 monomer. In another aspect thesecond cytokine is IL-10. In one embodiment, the second cytokine isIL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-21,IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons-α, -β, -γ, TGF-β,or tumor necrosis factors -α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, orIL-13. In a preferred embodiment, the second cytokine in the dualcytokine fusion protein comprising IL-10 and IL-2 or IL-4. In a morepreferred embodiment, the dual cytokine fusion protein is a sequence ofSEQ ID No: 35, 46−58 or 59. In yet another embodiment, the dual cytokinefusion protein will comprise an IL-10 variant molecule selected fromDV05, DV06, or DV07; the IL-10 variant molecule linked to a scaffoldingsystem comprising the VH and VL regions from a human anti-ebola antibody(i.e., Debo), wherein with the CDRs from an antibody selected from ananti-EGFR, anti-HER2, anti-CD14, anti-VEGFR1, anti-VEGFR2, anti-MAdCAM,or anti-PDGFR are engrafted into Debo; and a second cytokine selectedfrom IL-2, IL-4, IFNα is linked in the hinge region of the VH and VLpair. In a most preferred embodiment, the dual cytokine is a fusionprotein of SEQ ID No: 35, 46-58, or 59.

In still other embodiments, the dual cytokine fusion protein comprisingIL-10 incorporates linkers. A person of skill in the art knows thatlinkers or spacers are used to achieve proper spatial configuration ofthe various fusion protein parts and therefore may select theappropriate linker to use in the formation of the dual cytokine fusionprotein comprising IL-10. In a more preferred embodiment, the linker orspacer may be a random amino acid sequence (such as SSGGGGS (SEQ ID No.:39), GGGGSGGGGSGGGGS (SEQ ID No.: 40) or SSGGGGSGGGGSGGGGS (SEQ ID No.41)) a constant region of an antibody. The constant region can bederived from, but not limited to IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD,or IgE. In one embodiment, the linker or spacer is a constant heavy(“CH”) region 1, CH₂, or CH₃. In a more preferred embodiment, the linkeror spacer is a random amino acid sequence of SEQ ID No: 40. In anotheraspect, the linker or spacer may further comprise at least twointerchain disulfide bonds.

In other aspects, the present disclosure relates to nucleic acidmolecules that encode for the dual cytokine fusion protein comprisingIL-10 and a second cytokine. One embodiment therefore includes a nucleicacid sequence that encodes the protein set forth in SEQ ID No: 35,46-58, or 59. In a preferred embodiment, the nucleic acid sequenceincludes DK2¹⁰egfr (SEQ ID No: 60), DK2¹⁰her2 (SEQ ID No: 62 or 63),DK4¹⁰CD14DV06 or DK4¹⁰ngDV06CD14 (SEQ ID No: 61), or nucleic acidsequences that share 70% to 99% sequence homology thereof. In anotherembodiment, the nucleic acid sequence encodes a DK2¹⁰ form comprisingDV07 and targeting human VEGFR1 or VEGFR2; or to a molecule in DK4¹⁰form comprising DV06 and targeting human VEGFR1 or VEGFR2. Thepolynucleotide sequences that encode for the dual cytokine fusionprotein comprising IL-10 and a second cytokine may also includemodifications that do not alter the functional properties of thedescribed dual cytokine fusion protein. Such modifications will employconventional recombinant DNA techniques and methods. For example, theaddition or substitution of specific amino acid sequences may beintroduced into an IL-10 sequence at the nucleic acid (DNA) level usingsite-directed mutagenesis methods employing synthetic oligonucleotides,which methods are also well known in the art. In a preferred embodiment,the nucleic acid molecules encoding the dual cytokine fusion proteincomprising IL-10 and a second cytokine may include insertions,deletions, or substitutions (e.g., degenerate code) that do not alterthe functionality of the IL-10 variant molecule. The nucleotidesequences encoding the IL-10 variant and fusion proteins describedherein may differ from the amino acid sequences due to the degeneracy ofthe genetic code and may be 70-99%, preferably 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99%, homologous to the aforementioned sequences.Accordingly, an embodiment of the present disclosure includes a nucleicacid sequence that encodes a protein of SEQ ID Nos: 35, 46-58, or 59 butdiffering by 70-99% due to the degeneracy of the genetic code.

The nucleotide sequences encoding the dual cytokine fusion proteinsdescribed herein may further comprise well known sequences that aid in,for example, the expression, production, or secretion of the proteins.Such sequences may include, for example a leader sequence, signalpeptide, and/or translation initiation sites/sequence (e.g. Kozakconsensus sequence). The nucleotide sequences described herein may alsoinclude one of more restriction enzyme sites that allow for insertioninto various expression systems/vectors.

In another embodiment, the nucleotide sequences encoding the dualcytokine fusion protein may be used directly in gene therapy. In oneembodiment, the variant IL-10 molecules or fusion protein of the presentapplication can be delivered by any method know in the art, includingdirect administration of the mutant IL-10 protein and gene therapy witha vector encoding the mutant IL-10 protein. Gene therapy may beaccomplished using plasmid DNA or a viral vector, such as anadeno-associated virus vector, an adenovirus vector, a retroviralvector, etc. In some embodiments, the viral vectors of the applicationare administered as virus particles, and in others they are administeredas plasmids (e.g. as “naked” DNA).

Other methods for the delivery of the nucleotide sequences include thosewhich are already known in the art. These would include the delivery ofthe nucleotide sequences, such as but not limited to DNA, RNA, siRNA,mRNA, oligonucleotides, or variants thereof, encoding the IL-10 or IL-10variant molecules by a cell penetrating peptide, a hydrophobic moiety,an electrostatic complex, a liposome, a ligand, a liposomalnanoparticle, a lipoprotein (preferably HDL or LDL), a folate targetedliposome, an antibody (such as Folate receptor, transferrin receptor), atargeting peptide, or by an aptamer. The nucleotide sequences encodingIL-10 variant molecules may be delivered to a subject by directinjection, infusion, patches, bandages, mist or aerosol, or by thin filmdelivery. The nucleotide (or the protein) may be directed to any regionthat is desired for targeted delivery of a cytokine stimulus. Thesewould include, for example, the lung, the GI tract, the skin, liver,brain though intracranial injection, deep seated metastatic tumorlesions via ultrasound guided injections.

In another aspect, the present disclosure relates to methods ofpreparing and purifying the dual cytokine fusion protein comprisingIL-10. For example, nucleic acid sequences that encode the dual cytokinefusion protein described herein may be used to recombinantly produce thefusion proteins. For example, using conventional molecular biology andprotein expression techniques, the dual cytokine fusion proteindescribed herein may be expressed and purified from mammalian cellsystems. These systems include well known eukaryotic cell expressionvector systems and host cells. A variety of suitable expression vectorsmay be used and are well known to a person skilled in the art, which canbe used for expression and introduction of the variant IL-10 moleculesand fusion proteins. These vectors include, for example, pUC-typevectors, pBR-type vectors, pBI-type vectors, pGA-type, pBin19, pB1121,pGreen series, pCAMBRIA series, pPZP series, pPCV001, pGA482, pCLDO4541,pBIBAC series, pYLTAC series, pSB11, pSB1, pGPTV series, and viralvectors and the like can be used. Well known host cell systems includebut not limited to expression in CHO cells.

The expression vectors harboring the dual cytokine fusion protein mayalso include other vector componentry required for vector functionality.For example, the vector may include signal sequences, tag sequences,protease identification sequences, selection markers and other sequencesregulatory sequences, such as promoters, required for proper replicationand expression of the dual cytokine fusion protein. The particularpromoters utilized in the vector are not particularly limited as long asthey can drive the expression of the dual cytokine fusion protein in avariety of host cell types. Likewise, the type of Tag promoters are notbe limited as long as the Tag sequence makes for simpler or easierpurification of expressed variant IL-10 molecule easier. These mightinclude, for example, 6-histidine, GST, MBP, HAT, HN, S, TF, Trx, Nus,biotin, FLAG, myc, RCFP, GFP and the like can be used. Proteaserecognition sequences are not particularly limited, for instance,recognition sequences such as Factor Xa, Thrombin, HRV, 3C protease canbe used. Selected markers are not particularly limited as long as thesecan detect transformed rice plant cells, for example, neomycin-resistantgenes, kanamycin-resistant genes, hygromycin-resistant genes and thelike can be used.

The dual cytokine fusion protein described above may also includeadditional amino acid sequences that aid in the recovery or purificationof the fusion proteins during the manufacturing process. These mayinclude various sequence modifications or affinity tags, such as but notlimited to protein A, albumin-binding protein, alkaline phosphatase,FLAG epitope, galactose-binding protein, histidine tags, and any othertags that are well known in the art. See, e.g., Kimple et al (Curr.Protoc. Protein Sci., 2013, 73:Unit 9.9, Table 9.91, incorporated byreference in its entirety). In one aspect, the affinity tag is anhistidine tag having an amino acid sequence of HHHHHH (SEQ ID No.: 42).The histidine tag may be removed or left intact from the final product.In another embodiment, the affinity tag is a protein A modification thatis incorporated into the fusion protein (e.g., into the VH region of thefusion proteins described herein). A person of skill in the art willunderstand that any dual cytokine fusion protein sequence describedherein can be modified to incorporate a protein A modification byinserting amino acid point substitutions within the antibody frameworkregions as described in the art.

In another aspect, the protein and nucleic acid molecules encoding dualcytokine fusion protein may be formulated as a pharmaceuticalcomposition comprising a therapeutically effective amount of the dualcytokine fusion protein and a pharmaceutical carrier and/orpharmaceutically acceptable excipients. The pharmaceutical compositionmay be formulated with commonly used buffers, excipients, preservatives,stabilizers. The pharmaceutical compositions comprising the dualcytokine fusion protein is mixed with a pharmaceutically acceptablecarrier or excipient. Various pharmaceutical carriers are known in theart and may be used in the pharmaceutical composition. For example, thecarrier can be any compatible, non-toxic substance suitable fordelivering the dual cytokine fusion protein compositions of theapplication to a patient. Examples of suitable carriers include normalsaline, Ringer's solution, dextrose solution, and Hank's solution.Carriers may also include any poloxamers generally known to those ofskill in the art, including, but not limited to, those having molecularweights of 2900 (L64), 3400 (P65), 4200 (P84), 4600 (P85), 11,400 (F88),4950 (P103), 5900 (P104), 6500 (P105), 14,600 (F108), 5750 (P123), and12,600 (F127). Carriers may also include emulsifiers, including, but notlimited to, polysorbate 20, polysorbate 40, polysorbate 60, andpolysorbate 80, to name a few. Non-aqueous carriers such as fixed oilsand ethyl oleate may also be used. The carrier may also includeadditives such as substances that enhance isotonicity and chemicalstability, e.g., buffers and preservatives, see, e.g., Remington'sPharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, MackPublishing Company, Easton, Pa. (1984). Formulations of therapeutic anddiagnostic agents may be prepared by mixing with physiologicallyacceptable carriers, excipients, or stabilizers in the form oflyophilized powders, slurries, aqueous solutions or suspensions, forexample.

The pharmaceutical composition will be formulated for administration toa patient in a therapeutically effective amount sufficient to providethe desired therapeutic result. Preferably, such amount has minimalnegative side effects. In one embodiment, the amount of dual cytokinefusion protein administered will be sufficient to treat or preventinflammatory diseases or condition. In another embodiment, the amount ofdual cytokine fusion protein administered will be sufficient to treat orprevent immune diseases or disorders. Instill another embodiment, theamount of dual cytokine fusion protein administered will be sufficientto treat or prevent cancer. The amount administered may vary frompatient to patient and will need to be determined by considering thesubject's or patient's disease or condition, the overall health of thepatient, method of administration, the severity of side-effects, and thelike.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method route and dose of administration and the severity ofside effects. The appropriate dose administered to a patient istypically determined by a clinician using parameters or factors known orsuspected in the art to affect treatment or predicted to affecttreatment. Generally, the dose begins with an amount somewhat less thanthe optimum dose and it is increased by small increments thereafteruntil the desired or optimum effect is achieved relative to any negativeside effects. Important diagnostic measures include those of symptomsof, e.g., the inflammation or level of inflammatory cytokines produced.

The method for determining the dosing of the presently described dualcytokine fusion protein will be substantially similar to that describedin U.S. Pat. No. 10,858,412. Generally, the presently described dualcytokine fusion protein will have a dosing in the range of 0.5microgram/kilogram to 100 micrograms/kilogram. The dual cytokine fusionprotein may be administered daily, three times a week, twice a week,weekly, bimonthly, or monthly. An effective amount of therapeutic willimpact the level of inflammation or disease or condition by relievingthe symptom. For example, the impact might include a level of impactthat is at least 10%; at least 20%; at least about 30%; at least 40%; atleast 50%; or more such that the disease or condition is alleviated orfully treated.

Compositions of the application can be administered orally or injectedinto the body. Formulations for oral use can also include compounds tofurther protect the variant IL-10 molecules from proteases in thegastrointestinal tract. Injections are usually intramuscular,subcutaneous, intradermal or intravenous. Alternatively, intra-articularinjection or other routes could be used in appropriate circumstances.Parenterally administered dual cytokine fusion protein are preferablyformulated in a unit dosage injectable form (solution, suspension,emulsion) in association with a pharmaceutical carrier and/orpharmaceutically acceptable excipients. In other embodiments,compositions of the application may be introduced into a patient's bodyby implantable or injectable drug delivery system.

Testing the Dual Cytokine Fusion Protein

A plurality of screening assays are known and available to those ofskill in the art to test for the desired biological function. In oneembodiment, the desired biological function includes, but are notlimited to, reduced anti-inflammatory response, reduce T-cellstimulation, enhanced T-cell function, enhanced Kupffer cellfunctionality and reduced mast cell degranulation.

For example, it is known that IL-10 exposure primes T cells to generateand secrete more IFNγ upon T cell receptor stimulation. Simultaneously,IL-10 exposure prevents the secretion of TNFα, IL-6 and otherpro-inflammatory cytokines secreted from monocytes/macrophages inresponse to LPS. IL-10 also suppresses FoxP3⁺CD4⁺T_(reg)proliferation.In one embodiment, the dual cytokine fusion protein that maximizemonocyte/macrophage suppression but lack T cell effects, including bothstimulatory and suppressive responses, will be positively selected. Inone embodiment, screening for dual cytokine fusion proteins that possessincreased anti-inflammatory effects will be positively selected for thetreatment of autoimmune, anti-inflammatory disease or both. In anotherembodiments, dual cytokine fusion proteins that enhance Kupffer cellscavenging and lack T_(reg) suppression will also be selected to developfor treatment of Non-alcoholic Steatotic Hepatitis (NASH) and/orNon-alcoholic Fatty Liver Disease (NAFLD). In yet another embodiment,dual cytokine fusion proteins that maximize T cell biology, includingboth stimulatory and suppressive responses, and also possesses enhancedKupffer cell scavenging, will be selected to develop for the treatmentof cancer. Various assays and methods of screening the dual cytokinefusion proteins are previously described in co-pending U.S. Pat. No.10,858,412, which is incorporated by reference in its entirety. See,U.S. patent applcation Ser. No. 16/811,718 Specification at pages 39−42.

Methods of Treating and/or Preventing Using the Dual Cytokine

In other aspects, the present disclosure relates to methods of treatingand/or preventing malignant diseases or conditions or cancer comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the dual cytokine fusion protein comprising IL-10 and a secondcytokine. Such a protein will be in DK2¹⁰ form, where the fusion proteinwill comprise monomers of DV07 linked to a VH and VL scaffolding systemobtained from a human anti-ebola antibody which is engrafted with CDRsfrom any antibody targeting a tumor associated antigen (“TAA”); with asecond cytokine, IL-2, linked between the hinge region of the VH and VL.In a preferred embodiment, the dual cytokine fusion protein comprisesEBV IL-10 monomers of DV07. In a more preferred embodiment, the EBVIL-10 monomers include both substitutions at amino acid positions 31(V31L) and 75 (A751) of EBV IL-10 of SEQ ID NO: 3. In a more preferredembodiment, the EBV IL-10 is SEQ ID Nos: 11 or 16. In a preferredembodiment, the dual cytokine fusion protein comprises a VH and VL pairfrom an anti-ebola antibody, wherein the CDRs are substituted with 6CDRs from any TAA targeting antibody. In a preferred embodiment, the VHand VL regions of the dual cytokine fusion protein includes a VH of SEQID No: 37 and a VL of SEQ ID No: 38. In a more preferred embodiment, thedual cytokine fusion protein comprises a VH and VL pair from ananti-ebola antibody, wherein the CDRs are substituted with 6 CDRs from:an anti-EGFR antibody (SEQ ID Nos: 27, 29, 31, or 33), wherein thesecond cytokine is linked between the VH and VL regions of the scFv. Inother embodiments, the 6 CDR regions are substituted with 6 CDRs from ananti-Her2 Neu; an anti-PDGFR; anti-VEGFR1 and anti-VEGFR2, an anti-FGFR;an anti-HER3; or an anti-GPC3. Preferably the 6 CDRs are obtained fromanti-EGFR, or anti-HER2. In another preferred embodiment, the secondcytokine is an IL-2. In a most preferred embodiment, a dual cytokinefusion protein of SEQ ID Nos: 35 (EGFR targeting) or 52−55 (HER2targeting) is used to treat cancer.

In still other aspects, the present disclosure relates to methods oftreating and/or preventing inflammatory diseases or conditionscomprising administering to a subject in need thereof a therapeuticallyeffective amount of the dual cytokine fusion protein comprising IL-10(or variants thereof such as DV06) and a second cytokine (such as IL-4).In a preferred embodiment, the inflammatory diseases or disordersinclude, but are not limited to Crohn's disease, psoriasis, andrheumatoid arthritis (“RA”). Such a protein will be in DK4¹⁰ form, wherethe fusion protein will comprise monomers of DV06 linked to a VH and VLscaffolding system obtained from a human anti-ebola antibody which isengrafted with CDRs from any antibody targeting variousinflammatory/immune receptors or proteins (such as anti-CD14,anti-VEGFR2, anti-MAdCAM); with a second cytokine, IL-4 (SEQ ID No: 43)or a non-glycosylated form of IL-4 (SEQ ID No: 44), linked between thehinge region of the VH and VL. In an embodiment, the IL-10 monomerincludes wild type EBV IL-10, an EBV IL-10 variant with a single aminoacid substitution at position 75 of EBV IL-10 (DV06), or an EBV IL-10variant with two amino acid substitutions at positions 31 and 75 of EBVIL-10 (DV07). In a preferred embodiment, the EBV IL-10 monomers is wildtype EBV IL-10 or DV06. In a more preferred embodiment, the EBV IL-10 isSEQ ID Nos: 3, 9, 10, 11, 14 or 16. In a preferred embodiment, the dualcytokine fusion protein comprises a scaffolding system with a VH and VLpair from a human anti-ebola antibody. In a more preferred embodiment,the dual cytokine fusion protein used for treating inflammatory diseasesor conditions comprises a VH and VL pair from a human anti-ebolaantibody, wherein the CDRs are substituted with 6 CDRs from VH and VL ofan anti-MAdCAM antibody (preferably a human anti-MAdCAM antibody) or ananti-CD14 antibody (preferably a human anti-CD14 antibody) oranti-VEGFR2 (preferably a human anti-VEGFR2 antibody). In anotherpreferred embodiment, the second cytokine is an IL-4, preferably an IL-4variant having a N38A substitution (SEQ ID No. 44). In a most preferredembodiment, the inflammatory disease includes sepsis and/or septicshock, which is treated with a dual cytokine fusion protein comprisingDV06 or DV07 monomers and IL-4, wherein CDRs from an anti-CD14 antibodyare engrafted into an anti-ebola VH and VL scFv scaffolding system. In apreferred embodiment, the dual cytokine fusion protein is in DK4¹⁰ formof SEQ ID No: 56−58, or 59, more preferably SEQ ID No: 56. In anotherpreferred embodiment, the inflammatory disease includes IBD, which istreated with a dual cytokine fusion protein comprising DV06 monomers andIL-4 wherein the CDRs from an anti-MAdCAM antibody are engrafted into ananti-ebola VH and VLscFv scaffolding system. In yet another preferredembodiment, the inflammatory disease includes psoriasis or RA, which istreated with a dual cytokine fusion protein comprising DV06 monomers andIL-4 wherein the CDRs from an anti-VEGFR2 antibody are engrafted into ahuman anti-ebola VH and VL scFv scaffolding system. In a most preferredembodiment, a dual cytokine fusion protein of SEQ ID No: 46-50, 56-58,or 59 (CD14 targeting) or 51 (MAdCAM targeting) is used to reduceinflammation or sepsis.

In yet another aspect, the present disclosure relates to methods oftreating and/or preventing immune diseases or conditions comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the dual cytokine fusion protein comprising IL-10.

In other embodiments, the present disclosure also contemplates methodsof co-administration or treatment with a second therapeutic agent, e.g.,a cytokine, steroid, chemotherapeutic agent, antibiotic,anti-inflammatory agents, or radiation, are well known in the art. Thesemight include combination treatments with other therapeutic agents, suchas but not limited to one or more the following: chemotherapeutics,interferon-β, for example, IFNβ-1α and IFN-β-1β; a protein thatsimulates myelin basic protein; corticosteroids; IL-1 inhibitors; TNFinhibitors; anti-TNFα antibodies, anti-IL-6 antibodies, IL-1br-Igfusion, anti-IL-23 antibodies, antibodies to CD40 ligand and CD80;antagonists of IL-12 and IL-23, e.g., antagonists of a p40 subunit ofIL-12 and IL-23 (e.g., inhibitory antibodies against the p40 subunit);IL-22 antagonists; small molecule inhibitors, e.g., methotrexate,leflunomide, sirolimus (rapamycin) and analogs thereof, e.g., CCI-779;Cox-2 and cPLA2 inhibitors; NSAIDs; p38 inhibitors; TPL-2; Mk-2; NFkβinhibitors; RAGE or soluble RAGE; P-selectin or PSGL-1 inhibitors (e.g.,small molecule inhibitors, antibodies thereto, e.g., antibodies toP-selectin); estrogen receptor beta (ERB) agonists or ERB-NFkβantagonists.

Additionally, the combination treatment useful for administration withthe dual cytokine fusion protein may include TNF inhibitors include,e.g., chimeric, humanized, effectively human, human or in vitrogenerated antibodies, or antigen-binding fragments thereof, that bind toTNF; soluble fragments of a TNF receptor, e.g., p55 or β75 human TNFreceptor or derivatives thereof, e.g., 75 kdTNFR-IgG (75 kD TNFreceptor-IgG fusion protein, ENBREL™), p55 kD TNF receptor-IgG fusionprotein; and TNF enzyme antagonists, e.g., TNFα converting enzyme (TACE)inhibitors. Other combination treatment with anti-inflammatoryagents/drugs that includes, but not limited to standard non-steroidalanti-inflammatory drugs (NSAIDs) and cyclo-oxygenase-2 inhibitors. NSAIDmay include aspirin, celecoxib, diclofenac, diflunisal, etodolac,ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen,oxaprozin, piroxicam, salsalate, sulindac, and/or tolmetin. Thecyclo-oxygenase-2 inhibitor employed in compositions according to theapplication could, for example, be celecoxib or rofecoxib.

Additional therapeutic agents that can be co-administered and/orco-formulated with the dual cytokine fusion protein include one or moreof: interferon-β, for example, IFN β-1a and IFN β-1β; COPAXONE®;corticosteroids; IL-1 inhibitors; TNF antagonists (e.g., a solublefragment of a TNF receptor, e.g., p55 or β75 human TNF receptor orderivatives thereof, e.g., 75 kdTNFR-IgG; antibodies to CD40 ligand andCD80; and antagonists of IL-12 and/or IL-23, e.g., antagonists of a p40subunit of IL-12 and IL-23 (e.g., inhibitory antibodies that bind to thep40 subunit of IL-12 and IL-23); methotrexate, leflunomide, and asirolimus (rapamycin) or an analog thereof, e.g., CCI-779. Othertherapeutic agents may include Imfimzi or Atezolizumb.

For purposes of treating NASH, for example, the dual cytokine fusionprotein may be combined with cholesterol lowering agents, such asstatins and non-statin drugs. These agents include, but are not limitedto simvastatin, atorvastatin, rosuvastatin, lovastatin, pravastatin,gemfibrozil, fluvastatin, cholestyramine, fenofibrate, cholesterolabsorption inhibitors, bile acid-binding resins or sequestrants, and/ormicrosomal triglyceride transfer protein (MTP) inhibitors.

Representative chemotherapeutic agents that may be co-administered withthe dual cytokine fusion protein described herein may include forfollowing non-exhaustive list: include alkylating agents such asthiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime nitrogen mustardssuch as chiorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL® Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (Taxotere™, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; Xeloda® Roche, Switzerland; ibandronate; CPT11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoic acid; esperamicins; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogensincluding for example tamoxifen, raloxifene, aromatase inhibiting4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,onapristone, and toremifene (Fareston); and antiandrogens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

EXAMPLES Example 1: IL-10 and IL-2 Dual Cytokine Fusion Protein in VitroStudy

To evaluate the in vitro effects of targeting two cytokines to a tumor,a dual cytokine fusion protein, termed DK2¹⁰ (SEQ ID No: 35) (see FIG. 2as a representative diagram of the structure), was constructed from thefollowing components:

(a) two monomers of DV07 (which is a high affinity IL-10 receptorbinding, EBV IL-10 variant) coupled to a scFv with a VH and VL pairtargeting EGFR (the IL-10 fusion protein termed “SLP” of SEQ ID No. 31);and

(b) an IL-2 cytokine (SEQ ID No: 36);

where the IL-2 cytokine is conjugated or linked in the hinge (or linker)region between the VH (SEQ ID No: 37) and VL (SEQ ID No: 38) of the scFvtargeting EGFR (the SLP variant of SEQ ID No: 31).

This dual cytokine fusion protein was generated to evaluate the combinedeffects of these two cytokines on IL-2 induction of IFNγ from NK, CD4*and CD8* T cells. A comparative construct was also designed where theIL-2 was linked to the C-terminus of most C-terminal DV07 monomer of theSLP construct described above, creating a construct term “SLP-IL-2”(FIG. 3).

To test the effects of SLP-IL-2 (FIG. 3) and DK2¹⁰ (SEQ ID No: 35,schematically represented in FIG. 2) on the immune system, peripheralblood monocytes were isolated by magnetic bead positive selection toevaluate the DV07 function, and then NK, CD4* and CD8* T cells weresimilarly isolated for in vitro testing. A series of cellular in vitroassays were set up to model immunological function at different timepoints in the exposure cycle of a molecule injected subcutaneously inthe human body.

First, the effects of IL-10, IL-2, the combination of IL-10 and IL-2,and SLP-IL-2 were tested on monocytes/macrophages. This test shows thatIL-2 alone does not suppress TNFα, a proinflammatory cytokine, secretionin response to LPS, whereas the SLP:IL-2 construct, which comprises DV07was able to suppress proinflammatory cytokine secretion. A titration ofIL-10, IL-2, the combination of IL-10 and IL-2, and SLP-IL-2 wasperformed (FIG. 4). Unexpectedly, these data also suggest that thefunction of a DV07 containing construct is compromised by the additionof the IL-2 cytokine to the C-terminus of the IL-10 monomer (i.e.,SLP-IL-2; FIG. 3).

The effects of DK2¹⁰, which was designed as a DV07 containing variantwith IL-2 incorporated into the linker between the VH and VL of the scFvobtained from a human anti-ebola antibody, (schematically represented inFIG. 2), was also evaluated on monocytes/macrophages to determinewhether the construct retains IL-10 function. A titration of IL-10, SLP(an optimized variant of DegfrDV07 of SEQ ID No: 31), and DK2¹⁰egfr (SEDID No: 35) was performed (FIG. 5) and the data suggests that unlikelinking IL-2 to the C-terminus of the most C-terminal IL-10 monomer(SLP-IL-2), the unexpected incorporation of IL-2 into the linker betweenthe VH and VL of the scFv does not compromise the function of SLP (theDV07 containing IL-10 fusion protein of SEQ ID No: 31).

In order to assess the direct effects of DK2¹⁰egfr on T cells, an assaythat has been reported to directly elucidate the primary function ofIL-10 on CD8* T cells, predominantly the potentiation of IFNγ that isonly released upon T cell receptor engagement (Chan, 2015; Mumm J. ,2011; Emmerich, 2012) was performed.

The necessary therapeutic concentration of PEG-rHuIL-10 was found to be2−5 ng/mL, (Mumm J. , 2011; Naing A. , 2018; Naing A. , 2016) insystemic circulation. The CD8* T cell IFNγ assay exhibits maximal T cellIFNγ potentiation at 1-10 ng/mL, suggesting this is an appropriate modelassay system for evaluating the specific potency of IL-10 for cancerapplications.

High dose IL-2 therapy is the administration of between 600,000 to720,000 U/kg IL-2 every 8 hours for 5 days (Buchbinder, 2019) which isthe equivalent of 37-45 ug/kg, (1.1 mgs=18x106 IUs for IL-2). TheC_(max) concentration in systemic circulation for high dose IL-2 isbetween 37 to 45 ng/mL (Kirchner, 1998), where trough exposure is about10 ng/ml. These data suggest that the use of this assay is alsoappropriate for evaluating T cell response to IL-2 as maximal IL-2stimulation of antigen specific T cell function is approximately 10ng/ml in vitro. We therefore assessed the response of CD8* and CD4*T-cells to IL-10, IL-2, the combination of IL-10 and IL-2, SLP and DK2¹⁰in this assay format (FIG. 6). Unexpectedly, the tethering of IL-2 andDV07 together (i.e., tethering IL-2 to SLP in the into the linkerbetween the VH and VL of the scFv) increased the potency of eithermolecule alone by 100-fold (from ˜1-10 ng/mL to 0.01 ng/mL).Unexpectedly, the addition of untethered IL-2 and IL-10 at theseconcentrations did not enhance IFNγ secretion, which suggests that theeffect of tethering IL-2 and DV07 together leads to a significantlygreater than additive or synergistic effect on T cell function.

IL-2 toxicity (vascular leak syndrome) is associated with NK (Assier,2004), and CD4* T cell (Sivakumar, 2013), proinflammatory cytokinesecretion (Guan, 2007; Baluna, 1997). We therefore assessed whetherIL-10 could mute the proinflammatory effects of IL-2 on NK cellsdirectly isolated from blood. CD4* and CD8* T cells (1) directlyisolated from blood, (2) exposed to anti-CD3/anti-CD28 plus cytokines tomodel antigen priming, (3) exposed to cytokines after antigen priming tomodel exposure in the tumor and, (4) effect of exposure on antigenprimed T cell function upon engagement with cognate antigen (FIG. 7). NKcells, CD4* and CD8* T cells directly isolated from peripheral bloodwere treated with a titration of IL-10, IL-2, combination of IL-10 andIL-2, SLP (an optimized variant of DegfrDV07 of SEQ ID No: 31), andDK2¹⁰egfr for 4 days (FIG. 7). Expected dosing requirements forDK2¹⁰egfr is once every 4 days suggesting this in vitro exposure modelsa high concentration of cytokines (up to 100 ng/mL) for 4 days, farexceeding the expected C_(max) exposure. The data indicates that theaddition of IL-10 to IL-2 as individual cytokines or tether together asDK2¹⁰egfr suppresses IL-2 mediated induction of IFNγ secretion from NK,CD4* and CD8* T cells by ˜50%, ˜80% and ˜50% respectively at 5-10 ng/mL.At the expected therapeutic dose of 0.01 ng/mL, little to no IFNγ isinduced by the combined cytokines DK2¹⁰egfr.

The effect of cytokine exposure during model antigen presentation(immobilized 10 ng/mL anti-CD3/2 ng/mL anti-CD28), (Chan, 2015) was alsoexamined (FIG. 8). The data reveals that the addition of IL-10 to IL-2,and in particular the addition of tethered IL-2 and IL-10 via DK2¹⁰egfrsuppressed CD4* and CD8* IFNγ induction by ˜75% and ˜90% respectively at10 ng/mL and exhibits no IFNγ induction of 0.01 ng/mL.

Finally, the induction of IFNγ in CD4* and CD8* T cells after antigenexposure to model T cells trafficking in tumors prior to engagement withcognate tumor antigen was examined (FIG. 9). Unexpectedly, the datareveals that the effects of IL-2, IL-10 and IL-2 individually appliedversus DK2¹⁰egfr exert different functions on antigen primed CD4* andCD8* T cells. At expected therapeutic concentrations of DK2¹⁰egfr,DK2¹⁰egfr potentiates IFNγ secretion more than IL-2 or IL-10 and IL-2individually applied. At IL-10 and IL-2 expected therapeuticconcentrations, DK2¹⁰egfr, IL-2 and IL-10 and IL-2 individually appliedequivalently induce IFNγ secretion from CD4* and CD8* T cells. Thesedata collectively indicate the tethering of IL-2 and IL-10 (in the formof DK2¹⁰) together potentiate antigen specific CD4* and CD8* T cellresponses while suppressing pro-inflammatory cytokine secretionassociated with IL-2 toxicity. Notably, these effects were not impactedby the engraftment of the anti-EGFR CDRs into the anti-ebola scFvscaffolding.

Example 2: IL-10 and IL-2 Dual Cytokine Fusion Protein in Vivo Study

Targeting DV07 via an anti-EGFR scFv (wherein DV07 is fused to a scFvcomprising VH and VL obtained from a human anti-ebola ScFv scaffoldingcomprising 6 engrafted anti-EGFR CDRs; “Degfr:DV07” of SEQ ID No: 31)into the tumor microenvironment by virtue of generating a stablyexpressed human EGFR CT26 murine colorectal tumor cell line, waspreviously shown to exhibit superior anti-tumor function when comparedwith PEG-rHulL-10. See, U.S. Pat. No. 10,858,412. Using the same in vivotumor study, DK2¹⁰egfr was evaluated and compared to Degfr:DV07 in humanEGFR expressing CT26 cell murine tumor cell line.

CT26 (hEGFR*) tumor bearing B cell k.o. Balb/C mice, with an average of100 mm³ tumors were treated with test articles, doses and frequencies asprovided shown in Table 1. All test articles were administeredsubcutaneously in the scruff. All articles were dosed daily for 15 days.

TABLE 1 Test Articles, Doses and Frequencies No. Test article DoseFrequency 1 Vehicle 100 μl (control) Daily 2 Degfr:DV07 1 mg/kg Daily 3DK2¹⁰ 1 mg/kg Daily 4 DK2¹⁰ 2 mg/kg Daily 5 DK2¹⁰ 4 mg/kg DailyThe length and width of tumors were measured every three days byelectronic calipers and tumor volume was calculated ((LxW²)/2)). In thisexample, the terms “Degfr:DV07” is human EGFR targeted DV07; DK2¹⁰egfris abbreviated as “DK2¹⁰” and is human IL-2 coupled with DV07 via theCetuximab CDR grafted anti-ebola scFv scaffold. Methods

In vitro cell culture: CT26^((heGFR+)) tumor cells (ATCC) were grown to70% confluency in complete RPMI, 10% FCS, and 10ug/mL puromycin. Cellswere carried for no more than 3 passages in vitro prior to implantation.Cells were removed from cell culture plate using Accutase (Biolegend)and washed in complete RPMI spinning for 10 minutes at 400 g at 4° C.

Tumor Implantation: Tumor cells were implanted at 1×10⁵ cells/mouse in100 μL in 50% growth factor reduced Matrigel, 50% RPMI subcutaneous inthe right flank of B cell knockout mice.

Results

Comparison of Deqfr:DV07 and DK2¹⁰ on tumor growth: Targeting DV07 tothe tumor microenvironment via binding to the EGFR present on the stablytransfected tumor cells was previously show to be effective. See U.S.Pat. No. 10,858,412. Using the same tumor system, Degfr5DV07 versusDK2¹⁰was compared.

Tumors were measured three times a week (Table 2). Female Balb/C B cellknockout mice with 75 mm³ CT26(^(hEGFR)+) tumors were treatedsubcutaneously with the test articles and dosing frequencies illustratedin Table 2.

TABLE 2 Raw Data Group/ Days post Dosing Ear Dosing Day 0 Day 1 Day 3Day 6 Day 8 Day 10 Day 13 Day 15 Day 17 Animal # Tag # Material TVM TVMTVM TVM TVM TVM TVM TVM TVM D07-117-005 305 1. Vehicle 57 107 379 9211128 1664 D07-117-011 311 52 75 194 373 651 1211 D07-117-012 312 27 64108 247 578 1230 D07-117-013 313 33 152 407 542 725 1187 D07-117-014 31466 88 515 1274 1251 2461 47 97 321 671 867 1550 D07-117-003 303 2.DegfDV07 48 90 81 84 90 130 508 672 573 D07-117-006 306 1 mg/kg 62 105218 396 656 1195 1709 2291 3610 D07-117-007 307 56 80 122 131 215 333595 776 1008 D07-117-008 308 37 84 145 420 775 1124 2293 2850 2781D07-117-017 317 35 83 132 146 212 343 412 637 833 48 89 140 235 390 6251103 1445 1761 D07-117-001 301 3. DK2¹⁰ 57 107 286 478 638 927 1565 25672584 D07-117-004 304 1 mg/kg 55 183 241 192 145 392 735 788 1320D07-117-015 315 38 68 78 88 30 167 564 678 984 D07-117-018 318 54 103 7741 9 21 26 49 24 D07-117-020 320 38 65 45 0 0 0 0 0 0 48 105 145 160 164302 578 816 982 D07-117-024 324 4. DK2¹⁰ 69 116 57 9 0 0 0 0 0D07-117-029 329 2 mg/kg 40 87 134 34 52 135 361 391 624 D07-117-030 33032 37 141 96 118 339 641 912 1289 D07-117-031 331 66 83 68 0 0 0 0 0 0D07-117-039 339 32 64 117 239 439 878 1394 1675 2233 48 77 103 75 122271 479 596 829 D07-117-019 319 5. DK2¹⁰ 21 77 34 61 95 261 550 732 1127D07-117-032 332 4 mg/kg 56 111 34 0 0 0 0 0 0 D07-117-034 334 50 49 12549 27 0 0 0 0 D07-117-037 337 56 120 135 146 133 272 655 886 1413D07-117-038 338 59 114 74 63 36 97 270 380 553 48 94 80 64 58 126 295400 618

For this experiment, the CT26(^(hEGFR)+) cells were implanted at 1×10⁵cells in 50% growth factor reduced Matrigel to limit immunization of themice against tumor antigens.

The anti-tumor effect of Degfr:DV07 at 1 mg/kg was compared to the samedose of DK210 as well as 2 and 4 mg/kg doses (FIG. 10). 1 mg/kg dailydosing of DK2¹⁰ exerts superior anti-tumor function compared to 1 mg/kgdaily dosing of Degfr:DV07. 2 and 4 mg/kg doses of DK2¹⁰ exert moreanti-tumor function than 1 mg/kg.

Safety Assessment of DK2¹⁰: To test the safety of DK2¹⁰ dosing theweight of tumor bearing mice treated with Degfr:DV07 and DK2¹⁰ wasevaluated (FIG. 11). There are no apparent effects of dosing eitherDegfr:DV07 or DK2¹⁰ on the weight of the mice.

Effect of Degfr:DV07 and DK2¹⁰ dosinq on survival: The survivability ofCT26(^(hegf+)) tumor bearing mice to DK2¹⁰ was assessed (FIG. 12).

All tumors in the vehicle treatment mice were too large by IAACUCstipulation by day 17. 100%, 80%, 80% and 60% of mice were alive in the4 mg/kg, 2 mg/kg and 1 mg/kg DK210 and Degfr:DV07 1 mg/kg treatmentgroups at day 30 respectively.

These data collectively suggest coupling a high affinity IL-10 variant(DV07) to IL-2 and targeting both molecules to the tumormicroenvironment (via DK2¹⁰egfr) prevents overt IL-2 mediated toxicityat therapeutically effective doses. Engrafting anti-EGFR CDRs into thescFv scaffolding comprising VH and VL regions obtained from a humananti-ebola scaffolding does not impact the combined effects of IL-10 andIL-2, rather the anti-EGFR CDRs act as a means to concentrate the DK2¹⁰molecule in the tumor microenvironment. We believe that engrafting CDRsfrom any antibody (with appropriate optimization) that targets the tumormicroenvironment will result in the same or similar effect observed.

Example 3: IL-10 and IL-4 Dual Cytokine Fusion Protein

In Crohn's patients, high dose IL-10 led to diminished anti-inflammatoryresponses concomitant with increased IFNγ. To determine whethercombining a cytokine with IL-10 would enhanced the anti-inflammatoryfunction of IL-10 and suppress IL-10's stimulatory (IFNγ potentiation)function, IL-10 and IL-4 dual cytokine fusion proteins were generated.The inventor unexpectedly discovered that the combined treatment ofIL-10 and IL-4 on monocytes more potently suppressed LPS inducedinflammatory responses than either IL-V or IL-4 alone (discussed in moredetail below). In addition, IL-4 suppressed IL-10 mediated potentiationof IFNγ in CD8+T cells. Utilizing similar methods and rational fordesigning DK2V7egfr (described above in Examples 1 and 2), IL-4 orvarious IL-4 variants were coupled to IL-10 or IL-10 variants as afusion construct (see FIG. 17 as a representative diagram) to enhancethe suppressive function of IL-10. The resulting class of molecules wasa termed DK4¹⁰b

Table 3 provides a summary of the various molecules studied includingcytokines and various DK4¹⁰ fusion molecules.

TABLE 3 Tested Molecules Molecule Seq. ID No. Format Target rhIL-10 1Cytokine NA rhIL-4 43 Cytokine NA DeboDV06 21 Anti-ebola scaffoldcoupled to None monomers of DV06 DeboDV07 25 Anti-ebola scaffold coupledto None monomers DV07 DK4¹⁰DV06 46 Anti-ebola scaffold coupled to Nonewild type IL-4 and monomers of DV06 DK4¹⁰HADeglyDV06mCD14 47 Anti-ebolascaffold grafted Murine with anti-mCD14 CDR's CD14 coupled to the highaffinity, non-glycosylated IL-4 (T13D) and monomers of DV06DK4¹⁰HADeglyDV07mCD14 48 Anti-ebola scaffold grafted Murine withanti-mCD14 CDR's CD14 coupled to the high affinity, non-glycosylatedIL-4 (T13D) and monomers of DV07 DK4¹⁰ngDV06mCD14 49 Anti-ebola scaffoldgrafted Murine with anti-mCD14 CDR's CD14 coupled to the non-glycosylated IL-4 (N38A) and monomers of DV06 DK4¹⁰ngDV07mCD14 50Anti-ebola scaffold grafted Murine with anti-mCD14 CDR's CD14 coupled tothe non- glycosylated IL-4 (N38A) and monomers of DV07DK4¹⁰ngDV06mMAdCAM 51 Anti-ebola scaffold grafted Murine withanti-mMAdCAM CDR's MAdCAM coupled to the non- glycosylated IL-4 (N38A)and monomers of DV06The following molecules and combination of molecules were tested fortheir effects on monocyte/macrophages and CD8+T cells isolated bymagnetic bead positive selection, derived from peripheral bloodmononuclear cells (PBMC) preparations from healthy donors:

-   -   1. IL-4;    -   2. 1IL-10;    -   3. IL-4 in combination with IL-10;    -   4. DeboWtEBV;    -   5. DeboWtEBV in combination with IL-4;    -   6. DeboDV06;    -   7. DeboDV06 in combination with IL-4;    -   8. DeboDV07;    -   9. DeboDV07 in combination with IL-4;    -   10. DK4¹⁰ comprising wild type IL-4 and DV06 (“4DeboDV06”);    -   11. DK4¹⁰ comprising high affinity, non-glycosylated IL-4 (T13D)        and DV06 targeted to mCD14;    -   12. DK4¹⁰ comprising high affinity, non-glycosylated IL-4 (T13D)        and DV07 targeted to mCD14;    -   13. DK4¹⁰ comprising non-glycosylated IL-4 (N38A) with DV06        targeted to mCD14;    -   14. DK4¹⁰ comprising non-glycosylated IL-4 (N38A) with DV07        targeted to mCD14; and    -   15. DK4¹⁰ comprising non-glycosylated IL-4 with DV06 targeted to        mMAdCAM.

METHODS

PBMC and CD8+T-cell isolation: Both macrophages and CD8+T cells wereisolated from PBMC or leukopak using anti-CD14 (monocytes) or anti-CD8(CD8+T cells) magnetic microbeads by magnet assisted cell sorting.

Cellular Assay—Monocyte/Macrophage cell response to cytokines andlipopolysaccharide (LPS): In this assay, PMBC derived monocytes areisolated with CD14 positive selection beads, plated at 2×10⁵ cells/welland exposed to a titration cytokines and 10 ng/mL LPS. After 18 hours,supernatants are evaluated by ELISA for secreted proinflammatorycytokines. The percent reduction of TNFα is plotted to denote the effectthe cytokine or test article exerts on LPS. This assay mostappropriately mimics the response of monocytes to cytokines andbacterially derived proinflammatory products in peripheral blood.

Cellular Assay—CD8+T cells: Multiple CD8+T cells assays were used.Initially, CD8+T cells were derived from PBMC using CD8+positivemagnetic selection beads, plated at 2×105 cells/well and were exposed toa titration of cytokines or test articles under the followingconditions:

-   -   (i) 4 days alone,    -   (ii) 3 days to plate bound anti-CD3/anti-CD28 in the presence of        cytokines to mimic how these molecules affect the cells response        to cognate antigen presentation,    -   (iii) post anti-CD3/anti-CD28 for 3 days to mimic how antigen        stimulated cells respond to these cytokines and novel factors as        the cells enter the tumors, and    -   (iv) T cell receptor triggered IFNγ secretion was evaluated        after 4 hours from the cells exposed in vitro to mimic how T        cells in the tumor microenvironment respond to cognate antigen        exposure.

Both monocyte/macrophage and CD8+T cells were exposed to a titration ofhuman IL-4, IL-10, DeboWtEBV, DeboDV06 and the various DK4¹⁰ fusionmolecules at 0.1, 1, 10, 100 ng/mL or 0.001, 0.01, 0.1, 1 and 10 ng/mL(or molar equivalent) for overnight or 3−4 days as stated, with allconditions run in duplicate. Anti-inflammatory (monocytes/macrophages)and stimulatory effects (CD8+T cells) of these molecules were used todetermine the most effective anti-inflammatory pair of cytokines.

Protein measurements: Macrophage cell culture media was assayed by ELISAfor TNFα and CD8+T cell culture media was assayed by ELISA for IFNγ.DeboDV06, 4DeboDV06 and the various DK4¹⁰ fusion molecules were assessedby Nanodrop OD280 nM using each proteins' respective extinctioncoefficient and the concentration was corroborated by Coomassie stainedSDS-PAGE gel band intensity.

RESULTS

Development of Rational for IL-10 and IL-4 combination: IL-10 has beenreported to suppress TNFα secretion by macrophages in response to LPS(Malefyt, Interleukin 10 Inhibits Cytokine Synthesis by Human MonocytesAn Autoregulatory Role of IL-10 Produced by Monocytes, 1991; Moore,2001). IL-4 has been reported to suppress LPS induced TNFα secretionfrom human monocytes (Hart, Potential antiinflammatory effects ofinterleukin 4: Suppression of human monocyte tumor necrosis factor ca,interleukin 1, and prostaglandin E2, 1989) and human peritonealmacrophages (Hart, 1991).

To determine the effects of combining IL-4 and IL-10 on the suppressionof monocyte pro-inflammatory cytokine secretion in response to LPS as aninflammatory stimulus, peripheral blood monocytes were isolated fromhealthy donor PBMC by magnetic bead positive selection. The isolatedmonocytes were exposed to a titration of IL-10, IL-4, and a combinationof IL-10 and IL-4 (FIG. 13). Assessment of healthy human macrophageresponse to the titration, (0.1, 1, 10, 100 ng/mL) of human IL-10, IL-4,and the combination of IL-10 and IL-4 demonstrates that both IL-10 aloneand IL-4 alone are capable of suppressing LPS induced TNFα secretion.However, the combination of IL-10 and IL-4 together is superior insuppressing TNFα secretion to either cytokine alone.

Effect of IL-4 and DeboWtEBV on monocVte/macrophages: DeboWtEBV iscomprised of the wild type EBV IL-10 coupled to the half-life extendedVH and VL scaffolding system derived from a human anti-ebola antibody(previously described in U.S. Pat. No. 10,858,412). DeboWtEBV has beenshown to suppress TNFα secretion. The isolated monocytes were exposed toa titration of IL-10, IL-4, DeboWtEBV, and DeboWtEBV in combination withIL-4 (FIG. 14). The combination of IL-4 with DeboWtEBV together suppressLPS induced TNFα secretion from monocytes in a manner that is superiorto either IL-4 or DeboWtEBV alone.

Effect of IL-4 and DeboWtEBVon T cells: In addition to assessingcombined suppressive effects of IL-10 and IL-4 on monocyte/macrophages,the combined effects of IL-4 and DeboWtEBV on T cells were also examined(FIG. 15). DeboWtEBV induces less IFNγ from CD8+T cells compared to thesame molar concentration of IL-10. The combination of IL-4 withDeboWtEBV reduce IFNγ more than that induced by DeboWtEBV alone at 100ng/mL.

Effect of IL-4 and DeboDV06 on monocytes/macrophages: To determine ifthe suppressive effects of the IL-10 could be increased, a higheraffinity variant of the EBV IL-10, denoted as DV06 was assessed. DV06contains the point mutation (A751) and is coupled to the half-lifeextended VH and VL scaffolding system derived from a human anti-ebolaantibody (previously described in U.S. Pat. No. 10,858,412) bysubstituting wild type EBV IL-10 with DV06. Isolated monocytes wereexposed to a titration of IL-10, IL-4, DeboDV06, and DeboDV06 incombination with IL-4 (FIG. 16). DeboDV06 exhibits increased suppressivefunction relative to DeboWtEBV (compared with FIG. 15), and thecombination of DeboDV06 with IL-4 similarly increases the suppressivefunction on monocyte/macrophage response to LPS. The combination of IL-4with DeboDV06 suppress LPS induced TNFα secretion from monocytes in amanner that is superior to either IL-4 or DeboDV06 alone.

Evaluation of IL-4 coupled with DeboDV06 (in DK4¹⁰ form): The datasuggest that combining IL-4 with the IL-10 variant, DV06 (which is anenhanced affinity variant of wild type EBV IL-10), suppress LPS mediatedmonocyte inflammatory responses in a manner superior to either moleculealone. Accordingly, IL-4 was coupled to the DeboDV06 molecule byexpressing IL-4 in the linker between the VH and VL of the half-lifeextended scaffold molecule (FIG. 17), creating the first member of theDK4¹⁰ class of molecules denoted as “IL-4DeboDV06” or “4DeboDV06”, whichare non-targeting forms of the dual cytokine fusion protein (i.e.comprising the 6 CDR regions from the anti-ebola antibody).

Effect of IL-4DeboDV06 (in DK4¹⁰ form) on monocyte/macrophages: Todetermine whether IL-4DeboDV06, in DK4¹⁰ form, suppresses LPS inducedinflammatory responses, isolated monocytes were exposed to a titrationof IL-10, IL-4, DeboDV06, IL-10 in combination with IL-4, andIL-4DeboDV06 (FIG. 18). IL-4DeboDV06 in DK4¹⁰ form suppresses LPSinduced TNFα secretion from monocytes in a manner that is superior toeither IL-4 or DeboDV06 alone, but not quite as well as IL-4 plus IL-10,especially at lower concentrations.

Effect of IL-4DeboDV06 (in DK4¹⁰ form) on CD8+T cells: The ability ofIL-4DeboDV06 to potentiate and induce IFNγ from CD8+T cells was examinedand compared to IL-10, IL-4, DeboDV06, and DeboDV06 in combination withIL-4 (FIG. 19). IL-4DeboDV06 in DK4¹⁰ form suppresses IFNγ secretionfrom CD8+T cells similarly to the combination of DeboDV06 plus IL-4.

Effect of IL-4HADegIvDmCD14DV06 and IL-4HADegIvDmCD14DV07 (in DK4¹⁰form) on monocyte/macrophages: It was determined that the IL-4 aminoacid sequence used in manufacturing IL-4DeboDV06 in DK4¹⁰ form appearedto be glycosylated. Sequence analysis confirmed that a putative N-linkedglycosylation variant exists at amino acid position N38 but thatglycosylation is not required for function (Li, 2013). Further researchsuggested that substituting amino acid T13 with an aspartate (D)generated a high affinity IL-4 variant (U.S. Pat. No. 6,028,176). Bothpoint mutations with substitutions at N38A and T13D were introduced intoIL-4 and linked and incorporated into the Debo scaffolding engraftedwith 6 CDRs from murine CD14 (FIG. 20). The data suggests that the highaffinity, non-glycosylated IL-4 variant (i.e., comprising both the N38Aand T13D point mutations) exhibits inferior function in the DK4¹⁰coupled format when compared to wild type IL-4 in the same format.

Effect of IL-4ngDmCD14DV06 and IL-4ngDmCD14DV07 (in DK4¹⁰ form) onmonocyte/macrophages: The effects of IL-4ngDmCD14DV06 andIL-4ngDmCD14DV07 in DK4¹⁰ form, which includes an IL-4 variantcomprising the N38A substitution, were assessed by assaying for thesuppression of LPS induced inflammatory responses by exposing theisolated monocytes to a titration of IL-10, IL-4ngDmCD14DV06 (also knownas “DK4¹⁰mCD14DV06”) and IL-4ngDmCD14DV07 (also known as“DK4¹⁰mCD14DV07”) (FIG. 21). An IL-4 variant termed “IL-4ng” is thenon-glycosylated form of IL-4 (comprising the N38A substitution, SEQ IDNo: 44) that we introduced to improve manufacturability and “mCD14”represents the engraftment of the 6 CDRs from an anti-mCD14 antibodyinto the Debo scaffolding. Both DK4¹⁰ (comprising the IL-10 variants ofDV06 and DV07) molecules suppress LPS induced TNFα secretion.

Effect of IL-4ngDmCD14DV06 and IL-4ngDmCD14DV07 (in DK4¹⁰ form) on Tcells: The stimulatory effects of IL-10, IL-4ngDmCD14DV06 andIL-4ngDmCD14DV07 in DK4¹⁰ form (as described above) were assessed on Tcells (FIG. 22). Both DK4¹⁰ (comprising the IL-10 variants of DV06 andDV07) molecules do not induce as much IFNγ secretion as IL-10 from CD8+Tcells. IL-4ngDmCD14DV06 induces slightly less IFNγ secretion at 1-100 ngequivalent molar exposure in comparison to IL-4ngDmCD14DV07.

Effect of IL-4ngDmDMAdCAMDV06 (in DK4¹⁰ form) on monocyte/macrophages:The effects of IL-4ngDmMAdCAMDV06 in DK4¹⁰ form were assessed byassaying the suppression of LPS induced inflammatory response onmonocyctes/macrophages. IL-4ngDmMAdCAMDV06 is a dual cytokine fusion inDK4¹⁰ form comprising: (1) an IL-4ng variant that is non-glycosylated(comprising the N38A substitution); (2) the engraftment of the 6 CDRsfrom a mouse anti-MAdCAM antibody into the Debo scaffolding; and (3) theIL-10 variant DV06. Isolated monocytes/macrophages were titrated withIL-10 or IL-4ngDmMAdCAMDV06 (FIG. 23). IL-4ngDmMAdCAMDV06 suppresses LPSinduced TNFα secretion in monocytes/macrophages.

Effect of IL-4ngDmMAdCAMDV06 (DK4⁰ format) on T cells: We also evaluatedthe stimulatory effects of IL-10 and IL-4ngDmMAdCAMDV06 (DK4¹⁰ format)on T cells (FIG. 24). IL-4ngDmMAdCAMDV06 does not induce IFNγ secretionfrom CD8+T cells unlike IL-10.

CONCLUSION

These data suggest that IL-4 variants and IL-10 variants can beco-expressed via coupling these two cytokines to a human anti-eboladerived VH/VL scaffold system (i.e., in DK4¹⁰ form). The combination ofIL-4 and IL-10 variants suppresses LPS induced inflammatory responses bymonocyte/macrophages while also inhibiting the induction of IFNγ fromCD8+T cells, regardless of the targeting CDR present within the VH andVL scaffolding system (compare 4DeboDV06 to engrafted versions of DK4¹⁰comprising CDRs from anti-mCD14 and anti-mMAdCAM).

The anti-ebola derived VH and VL scaffold couples IL-4 and IL-10 variantcytokines effectively and can accept multiple targeting CDR's grafts.The combination of IL-4ng (the IL-4 variant resulting innon-glycosylated IL-4 due to the N38A substitution) with DV06 suppressesLPS mediated TNFα secretion effectively from 0.1-100 ngs/mL and does notinduce significant IFNγ from CD8+T cells in the same dose range.

Example 4: DK4¹⁰ in the Treatment of Sepsis

Having determined that IL-4ngDmCD14DV06 (also known as “DK4¹⁰mCD14DV06”)was capable of suppressing LPS induced TNFα secretion and tamped downthe induction of IFNγ from CD8+T-cells (see, FIG. 21 and FIG. 22), thismolecule was examined in a well-known and conventional sepsis model.

Briefly, wild type Balb/C mice were obtained and acclimated, pursuantstandard IACUCU protocols. The mice were maintained on standard chow andwater ad libitum with a 12 hour light/dark cycle.

Vehicle, DK4¹⁰mCD14DV06, was dosed subcutaneously in the animal at thestated dose in 100 milliliters of vehicle buffer at the stated timepoints either before (“pre”) or after (“post”) intraperitoneal LPSadministration (350 mg/mouse).

After 4 days of acclimation, five (5) mice per group were treated withthe following:

-   -   (1) 1 mg/kg DK410mCD14DV06 30 minutes before LPS administration;        and    -   (2) 1 mg/kg DK410mCD14DV06 30 minutes after LPS administration

The mice were evaluated for survival 48 hours after LPS administration.Treatment of mice with DK410mCD14DV06 30 minutes before LPSadministration resulted in 100% survivor rate, whereas treatment withDK410mCD14DV06 30 minutes after LPS administration demonstratedprotective effects against septic shock (FIG. 25).

The data suggests that coupling an IL-10 variant to an IL-4 variant(IL-4ng) and targeting the two molecules via a Debo scaffolding systemwith 6 CDRs from a mouse anti-CD14 antibody (e.g., using DK4¹⁰mCD14DV06)significantly attenuates the inflammatory response and treats septicshock.

This written description uses examples to disclose aspects of thepresent disclosure, including the preferred embodiments, and also toenable any person skilled in the art to practice the aspects thereof,including making and using any devices or systems and performing anyincorporated methods. The patentable scope of these aspects is definedby the claims, and may include other examples that occur to thoseskilled in the art. Such other examples are intended to be within thescope of the claims if they have structural elements that do not differfrom the literal language of the claims, or if they include equivalentstructural elements with insubstantial differences from the literallanguage of the claims. Aspects from the various embodiments described,as well as other known equivalents for each such aspect, can be mixedand matched by one of ordinary skill in the art to construct additionalembodiments and techniques in accordance with principles of thisapplication.

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1. A dual cytokine fusion protein of formula (I)NH₂-(IL-10)—(X1)—(Z_(n))—(X2)—(IL-10)—COOH  (Formula I); wherein “IL-10”is a monomer; “X1” is a VL or VH region from a first monoclonalantibody; “X2” is a VH or VL region from the first monoclonal antibody;wherein when X1 is a VL, X2 is a VH or when X1 is a VH, X2 is a VL,wherein the first monoclonal antibody is an anti-ebola antibody; whereinthe VL and VH from the anti-ebola antibody include 3 light chain CDRsand 3 heavy chain CDRs that are engrafted with 3 light chain CDRs and 3heavy chain CDRs from a second monoclonal “Z” is a cytokine other thanIL-10; “n” is an integer of 1; and wherein: i. the IL-10 is SEQ ID No:1, the second antibody is an anti-epidermal growth factor receptor(EGFR) monoclonal antibody, and Z is IL-2; ii. the IL-10 is SEQ ID No:1, the second antibody is an anti-EGFR monoclonal antibody, and Z isIL-15; iii. the IL-10 is SEQ ID No: 1, the second antibody is ananti-EGFR monoclonal antibody, and Z is IL-7; iv. the IL-10 is SEQ IDNo: 1, the second antibody is an anti-EGFR monoclonal antibody, and Z isIL-28; v. the IL-10 is SEQ ID No: 1, the second antibody is an anti-EGFRmonoclonal antibody, and Z is IL-29; vi. the IL-10 is SEQ ID No: 1, thesecond antibody is an anti-HER2 monoclonal antibody, and Z is IL-2; vii.the IL-10 is SEQ ID No: 1, the second antibody is an anti-HER2monoclonal antibody, and Z is IL-15; viii. the IL-10 is SEQ ID No: 1,the second antibody is an anti-HER2 monoclonal antibody, and Z is IL-7;ix. the IL-10 is SEQ ID No: 1, the second antibody is an anti-HER2monoclonal antibody, and Z is IL-28; x. the IL-10 is SEQ ID No: 1, thesecond antibody is an anti-HER2 monoclonal antibody, and Z is IL-29; xi.the IL-10 is SEQ ID No: 1, the second antibody is an anti-VEGFR2monoclonal antibody, and Z is IL-2; xii. the IL-10 is SEQ ID No: 1, thesecond antibody is an anti-VEGFR2 monoclonal antibody, and Z is IL-15;xiii. the IL-10 is SEQ ID No: 1, the second antibody is an anti-VEGFR2monoclonal antibody, and Z is IL-7; xiv. the IL-10 is SEQ ID No: 1, thesecond antibody is an anti-VEGFR2 monoclonal antibody, and Z is IL-28;xv. the IL-10 is SEQ ID No: 1, the second antibody is an anti-VEGFR2monoclonal antibody, and Z is IL-29; xvi. the IL-10 is SEQ ID No: 1, thesecond antibody is an anti-PDGFR monoclonal antibody, and Z is IL-2;xvii. the IL-10 is SEQ ID No: 1, the second antibody is an anti-PDGFRmonoclonal antibody, and Z is IL-15; xviii. the IL-10 is SEQ ID No: 1,the second antibody is an anti-PDGFR monoclonal antibody, and Z is IL-7;xix. the IL-10 is SEQ ID No: 1, the second antibody is an anti-PDGFRmonoclonal antibody, and Z is IL-28; xx. the IL-10 is SEQ ID No: 1, thesecond antibody is an anti-PDGFR monoclonal antibody, and Z is IL-29;xxi. the IL-10 is SEQ ID No: 1, the second antibody is an anti-GPC3monoclonal antibody, and Z is IL-2; xxii. the IL-10 is SEQ ID No: 1, thesecond antibody is an anti-GPC3 monoclonal antibody, and Z is IL-15;xxiii. the IL-10 is SEQ ID No: 1, the second antibody is an anti-GPC3monoclonal antibody, and Z is IL-7; xxiv. the IL-10 is SEQ ID No: 1, thesecond antibody is an anti-GPC3 monoclonal antibody, and Z is IL-28;xxv. the IL-10 is SEQ ID No: 1, the second antibody is an anti-GPC3monoclonal antibody, and Z is IL-29; xxvi. the IL-10 is SEQ ID No: 1,the second antibody is an anti-PD-L1 monoclonal antibody, and Z is IL-2;xxvii. the IL-10 is SEQ ID No: 1, the second antibody is an anti-PD-L1monoclonal antibody, and Z is IL-15; xxviii. the IL-10 is SEQ ID No: 1,the second antibody is an anti-PD-L1 monoclonal antibody, and Z is IL-7;xxix. the IL-10 is SEQ ID No: 1, the second antibody is an anti-PD-L1monoclonal antibody, and Z is IL-28; xxx. the IL-10 is SEQ ID No: 1, thesecond antibody is an anti-PD-L1 monoclonal antibody, and Z is IL-29;xxxi. the IL-10 is SEQ ID No: 3, the second antibody is an anti-CD14monoclonal antibody, and Z is IL-4; xxxii. the IL-10 is SEQ ID No: 3,the second antibody is an anti-MAdCAM monoclonal antibody, and Z isIL-4; xxxiii. the IL-10 is SEQ ID No: 3, the second antibody is ananti-VEGFR2 monoclonal antibody, and Z is IL-4; xxxiv. the IL-10 is SEQID No: 14, the second antibody is an anti-CD14 monoclonal antibody, andZ is IL-4; xxxv. the IL-10 is SEQ ID No: 14, the second antibody is ananti-MAdCAM monoclonal antibody, and Z is IL-4; xxxvi. the IL-10 is SEQID No: 14, the second antibody is an anti-VEGFR2 monoclonal antibody,and Z is IL-4; xxxvii. the IL-10 is SEQ ID No: 16, the second antibodyis an anti-EGFR monoclonal antibody, and Z is IL-2; xxxviii. the IL-10is SEQ ID No: 16, the second antibody is an anti-EGFR monoclonalantibody, and Z is IL-15; xxxix. the IL-10 is SEQ ID No: 16, the secondantibody is an anti-EGFR monoclonal antibody, and Z is IL-7; xl. theIL-10 is SEQ ID No: 16, the second antibody is an anti-EGFR monoclonalantibody, and Z is IL-28; xli. the IL-10 is SEQ ID No: 16, the secondantibody is an anti-EGFR monoclonal antibody, and Z is IL-29; xlii. theIL-10 is SEQ ID No: 16, the second antibody is an anti-HER2 monoclonalantibody, and Z is IL-2; xliii. the IL-10 is SEQ ID No: 16, the secondantibody is an anti-HER2 monoclonal antibody, and Z is IL-15; xliv. theIL-10 is SEQ ID No: 16, the second antibody is an anti-HER2 monoclonalantibody, and Z is IL-7; xlv. the IL-10 is SEQ ID No: 16, the secondantibody is an anti-HER2 monoclonal antibody, and Z is IL-28; xlvi. theIL-10 is SEQ ID No: 16, the second antibody is an anti-HER2 monoclonalantibody, and Z is IL-29; xlvii. the IL-10 is SEQ ID No: 16, the secondantibody is an anti-VEGFR2 monoclonal antibody, and Z is IL-2; xlviii.the IL-10 is SEQ ID No: 16, the second antibody is an anti-VEGFR2monoclonal antibody, and Z is IL-15; xlix. the IL-10 is SEQ ID No: 16,the second antibody is an anti-VEGFR2 monoclonal antibody, and Z isIL-7; I. the IL-10 is SEQ ID No: 16, the second antibody is ananti-VEGFR2 monoclonal antibody, and Z is IL-28; Ii. the IL-10 is SEQ IDNo: 16, the second antibody is an anti-VEGFR2 monoclonal antibody, and Zis IL-29; lii. the IL-10 is SEQ ID No: 16, the second antibody is ananti-PDGFR monoclonal antibody, and Z is IL-2; liii. the IL-10 is SEQ IDNo: 16, the second antibody is an anti-PDGFR monoclonal antibody, and Zis IL-15; liv. the IL-10 is SEQ ID No: 16, the second antibody is ananti-PDGFR monoclonal antibody, and Z is IL-7; Iv. the IL-10 is SEQ IDNo: 16, the second antibody is an anti-PDGFR monoclonal antibody, and Zis IL-28; Ivi. the IL-10 is SEQ ID No: 16, the second antibody is ananti-PDGFR monoclonal antibody, and Z is IL-29; Ivii. the IL-10 is SEQID No: 16, the second antibody is an anti-GPC3 monoclonal antibody, andZ is IL-2; Iviii. the IL-10 is SEQ ID No: 16, the second antibody is ananti-GPC3 monoclonal antibody, and Z is IL-15; Iix. the IL-10 is SEQ IDNo: 16, the second antibody is an anti-GPC3 monoclonal antibody, and Zis IL-7; Ix. the IL-10 is SEQ ID No: 16, the second antibody is ananti-GPC3 monoclonal antibody, and Z is IL-28; Ixi. the IL-10 is SEQ IDNo: 16, the second antibody is an anti-GPC3 monoclonal antibody, and Zis IL-29; Ixii. the IL-10 is SEQ ID No: 16, the second antibody is ananti-PD-L1 monoclonal antibody, and Z is IL-2; Ixiii. the IL-10 is SEQID No: 16, the second antibody is an anti-PD-L1 monoclonal antibody, andZ is IL-15; Ixiv. the IL-10 is SEQ ID No: 16, the second antibody is ananti-PD-L1 monoclonal antibody, and Z is IL-7; Ixv. the IL-10 is SEQ IDNo: 16, the second antibody is an anti-PD-L1 monoclonal antibody, and Zis IL-28; or Ixvi. the IL-10 is SEQ ID No: 16, the second antibody is ananti-PD-L1 monoclonal antibody, and Z is IL-29.
 2. The dual cytokinefusion protein according to claim 1, wherein xxxiv. is a fusion proteinof SEQ ID No: 47, 48, 49, 50, 56, 57, 58, or
 59. 3. The dual cytokinefusion protein according to claim 1, wherein xxxv. is a fusion proteinof SEQ ID NO:
 51. 4. The dual cytokine fusion protein according to claim1, wherein xxxvii. is a fusion protein of SEQ ID No:
 35. 5. The dualcytokine fusion protein according to claim 1, wherein xlii. is a fusionprotein of SEQ ID No: 52, 53, 54, or
 55. 6. A dual cytokine fusionprotein of SEQ. ID No:
 46. 7. A pharmaceutical composition comprisingthe dual cytokine fusion protein according to claim 1, pharmaceuticallyacceptable buffers, and pharmaceutically excipients, wherein the dualcytokine fusion protein is any one of i. to Ixv. or Ixvi.
 8. Apharmaceutical composition comprising the dual cytokine fusion proteinaccording to claim 7, pharmaceutically acceptable buffers, andpharmaceutically excipients.
 9. A pharmaceutical composition comprisingthe dual cytokine fusion protein according to claim 6, pharmaceuticallyacceptable buffers, and pharmaceutically excipients.
 10. Apharmaceutical composition comprising the dual cytokine fusion proteinaccording to claim 9, pharmaceutically acceptable buffers, andpharmaceutically excipients.
 11. A method of treating cancer comprisingadministering to a subject in need thereof, an effective amount of acomposition comprising the dual cytokine fusion protein according toclaim 1, wherein the dual cytokine fusion protein is any one of i. toxxx, xxxvii. to Ixv. or Ixvi.
 12. The method according to claim 9,wherein the dual cytokine fusion protein is xxxvii.
 13. The methodaccording to claim 12, wherein xxxvii. is SEQ ID No:
 35. 14. The methodaccording to claim 11, wherein the dual cytokine fusion protein is xlii.15. The method according to claim 14, wherein xlii. is SEQ ID No: 52,53, 54, or
 55. 16. A method of treating inflammatory bowel disease (IBD)comprising administering to a subject in need thereof, an effectiveamount of a composition comprising the dual cytokine fusion proteinaccording to claim 1, wherein the dual cytokine fusion protein is xxxii.or xxxv.
 17. The method according to claim 16, wherein the IBD isCrohn's disease or ulcerative colitis.
 18. The method according to claim16, wherein xxxv. is SEQ ID No:
 51. 19. A method of treating sepsis,septic shock, and/or symptoms associated thereof comprisingadministering to a subject in need thereof, an effective amount of acomposition comprising the fusion protein according to claim 1, whereinthe dual cytokine fusion protein is xxxi. or xxxiv.
 20. The methodaccording to claim 19, wherein the xxxiv. is SEQ ID No: 47, 48, 49, 50,56, 57, 58, or
 59. 21. A method of treating psoriasis or rheumatoidarthritis comprising administering to a subject in need thereof, aneffective amount of a composition comprising the dual cytokine fusionprotein according to claim 1, wherein the dual cytokine fusion proteinis xxxiii. or xxxvi.